WO2006075964A1 - Procede permettant de co-transporter un reactif avec une substance macromoleculaire amphiphile dans un conduit de transport microfluidique - Google Patents

Procede permettant de co-transporter un reactif avec une substance macromoleculaire amphiphile dans un conduit de transport microfluidique Download PDF

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Publication number
WO2006075964A1
WO2006075964A1 PCT/SE2006/000070 SE2006000070W WO2006075964A1 WO 2006075964 A1 WO2006075964 A1 WO 2006075964A1 SE 2006000070 W SE2006000070 W SE 2006000070W WO 2006075964 A1 WO2006075964 A1 WO 2006075964A1
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WIPO (PCT)
Prior art keywords
reactant
liquid
protocol
transporting
typically
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PCT/SE2006/000070
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English (en)
Inventor
Mats INGANÄS
Rikard KÅNGE
Magnus Gustafsson
Johan ENGSTRÖM
Bo Ek
Helene Derand
Ann-Kristin Honerud
Original Assignee
Gyros Patent Ab
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Priority claimed from US11/038,712 external-priority patent/US8592219B2/en
Application filed by Gyros Patent Ab filed Critical Gyros Patent Ab
Priority to JP2007551224A priority Critical patent/JP2008527371A/ja
Priority to EP06700622A priority patent/EP1848996A4/fr
Publication of WO2006075964A1 publication Critical patent/WO2006075964A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B01L2300/163—Biocompatibility

Definitions

  • the transportation is typically part of a predetermined protocol for processing the aliquots.
  • the protocol may be preparative, analytical, synthetic, etc.
  • At least one of the aliquots contains a reactant used in the protocol while other aliquots may be devoid of such reactants, e.g., by merely functioning as washing liquids and/or conditioning liquids and/or diluents for aliquots containing a reactant.
  • analytes and other reactants used in microfluidic protocols need special precautions in order for a given protocol to reach an acceptable detection limit, precision, recovery etc., and/or to avoid loss of analyte due to undesired interactions with inner surfaces. It is attractive to explain at least a part of these difficulties in terms of deficiencies in the surface modification methods and/or the material in which the enclosed microchannel structures are fabricated.
  • the problems encountered typically depend on type of analyte, matrix in which the analyte occurs (serum, plasma, urine, culture supernatant etc.), kind of assay, kind of device including inner surfaces of the device etc.
  • Problematic reactants, in particular analytes typically are bioorganic molecules.
  • An embodiment of the present invention comprises improved compositions, agents and/or methods that generically could be used for improving detection limits and/or precision and/or recovery and/or to prevent loss of analyte in micro fluidic protocols of the kind discussed above.
  • the improvement in detection limit for microfluidic assay protocols typically includes measurement of analytes that occur at least down to 10 "6 mole/1, such as down 10 " mole/1 or down to 10 "12 mole/1 or down to 10 "15 mole/1 in a native biological fluid.
  • the required detection limit for an analyte is as a rule always > 10 "25 mole/1.
  • the improvement in precision typically means that levels of the analyte concerned is obtained with a coefficient of variation (CV) that is with ⁇ 20%, such as ⁇ 15% or + 10% or + 5%.
  • CV coefficient of variation
  • the improvement in recovery typically means that recovery-values > 60%, such as > 75% or > 90% or > 95% or essentially around 100% are accomplished.
  • compositions and/or methods that could be used for reducing undesired interactions between constituents of liquids used in microfluidic protocols and/or between such constituents and inner surfaces of the devices used, in particular in transport microconduits.
  • Typical interactions to be reduced include fouling, e.g., undesired adsorption and denaturation caused by contact with inner surfaces.
  • compositions and methods that enable minimizing matrix effects in the experimental protocols discussed in the context of the invention.
  • the present invention can enable use of pre-prepared standard curves and standard values in microfluidic assays protocols, e.g., standard curves/values that are applicable to measured values that are obtained a) at different dates for different analyte samples, and/or b) for two or more different kinds of analyte sample matrixes, e.g., selected from serum, plasma, urine, lachrymal fluid, semen, regurgitated fluid, lymphae, cerebrospinal fluid, cell or tissue homogenates of different origins, celljculture supernatant etc.
  • analyte sample matrixes e.g., selected from serum, plasma, urine, lachrymal fluid, semen, regurgitated fluid, lymphae, cerebrospinal fluid, cell or tissue homogenates of different origins, celljculture supernatant etc.
  • the present inventors have found that it can be easier to obtain acceptable results for problematic microfluidic protocols if an exogenous amphiphilic macromolecular substance that typically is capable of forming micelles in water is co-transported with one or more of the liquids transported when performing a given protocol in a microfluidic device, i.e. the substance is present in with the liquids.
  • Typical examples of this kind of substances are milk protein, such as ⁇ -casein and other caseins.
  • Beneficial effects have been obtained both when the transport is taking place within a microfluidic device or in a transport microconduit of a separate device used for the dispensation of liquid to the microfluidic device.
  • the liquids concerned typically contain a reactant, such as an analyte, or are priming, washing or conditioning liquids (WC-liquids).
  • a reactant such as an analyte
  • WC-liquids priming, washing or conditioning liquids
  • exogenous means that the substance is not natively present in a biologically derived sample that is used in the protocol to be carried out.
  • An embodiment of the present invention comprises a method of transporting an aliquot of liquid (aliquot/liquid] . ), comprising the steps of providing the aliquot within an upstream end of the microconduit (transport microconduit), and applying a driving force for transporting the aliquot through the microconduit.
  • the aliquot may or may not contain a reactant which is dissolved in the aliquot/liquid and needed for the experimental protocol performed.
  • liquidi/aliquoti contains one or more of certain protocol enhancers as discussed below that thus are co-transported with the liquid/aliquotand any reactants present therein.
  • the inner surface of the transport microconduit may be coated with the enhancer before the transport of a liquid aliquot that may or may not contain a protocol enhancer of the types discussed below.
  • the driving force used for liquid transport depend on the device in which the transport microconduit is present. Appropriate forces may be created actively, for instance by applying a pressure differential over the microconduit or by passive means. Transport forces may be created electrokinetically e.g., electroendosmosis, or non-electrokinetically e.g. capillary force, inertia force such as centrifugal force, hydrostatic force, forces created by different kinds of pumps etc. See the discussion about dispensation devices and microfluidic devices.
  • the liquid is typically aqueous, for instance with water as the main liquid component (> 30 %, such as > 50% (v/v)) possibly in admixture with one or more water-miscible organic liquid components.
  • the protocol enhancer is an amphiphilic macromolecular substance that typically is capable of forming micelles (micelle- forming) with itself in water if present above a certain concentration, and/or that inner surfaces of the microconduit are coated with such a macromolecular substance. Presence of the substance together with a reactant, such as an analyte or a reagent, in a liquid aliquot to be transported means that the two entities are co-transported.
  • liquidi contains an immunoglobulin preparation (Ig) and/or that inner surfaces of the microconduit is coated with such a preparation.
  • Ig immunoglobulin preparation
  • This subaspect is typically used in analytical protocols that comprise assaying for an analyte in a biological sample by the use of an antibody reagent.
  • a reactant such as an analyte or a reagent
  • this subaspect means co-tranporting the Ig together with the reactant.
  • the characteristic feature is that UqUId 1 has an increased concentration of salt (ionic strength) and/or a surfactant that is different from the macromolecular substance of the 1st subaspect.
  • This subaspect is typically used in analytical protocols that comprise assaying for an analyte that carries one or more groups that are charged at the pH of liquidi and/or one or more hydrophobic groups.
  • a reactant such as an analyte or a reagent
  • this subaspect means co-tranporting the salt and/or this kind of surfactant with the reactant.
  • Each of the enhancers given above for the lst-3rd subaspects may be combined with one or more other protocol enhancers.
  • the protocol enhancer of the 1st subaspect is of utmost importance in the invention, but .not imperative.
  • a process protocol utilizing the inventive concept typically comprises processing one or more liquid aliquots through the same transport microconduit or through different transport microconduits that either is/are present in a microchannel structure of a microfluidic device or in a device for dispensation of liquid to a microdevice. At least one of these aliquots has one or more of the characteristic features outlined herein for a liquid aliquot to be transported, e.g. contains a reactant of the protocol contemplated and/or a protocol enhancer, e.g. according to one or more of the 1st - 3rd subaspects, such as the amphiphilic macromolecular substance. If the protocol is analytical (e.g.
  • the reactant in one or more of the aliquots may be an analyte or a reagent. If an aliquot of liquid contains a reactant, this reactant is typically different from the reactant of other aliquots.
  • One or more of the aliquots transported may have been produced within the microchannel structure but derive from one, two or more aliquots that have been dispensed in one or more earlier steps to the structure, e.g. produced by performing a reaction, a separation, an adsorption, a desorption, a mixing, a diluting etc involving the earlier introduced aliquots. This applies to aliquots containing a reactant as well as to aliquots not containing a reactant.
  • Figure 1 illustrates the effect of the invention on an hIL-8 assay.
  • Figure 2 illustrates a comparison between a conventional diluent and a diluent according to the invention.
  • Figure 3 concludes screening experiments with different combinations of protocol enhancers diluents for diluting five samples containing different analytes (mTNF ⁇ , mIL-6, hTNF ⁇ , hIL-8, hMCP-1, hlFN ⁇ )
  • microfluidic device refers to a device having one or a plurality of microchannel structures which each contains all the functionalities that are required for carrying out those parts of a given microfluidic protocol that are to be carried out in the microfluidic device.
  • Microfluidic protocols and microfluidic devices have been described by among others by Gyros AB/Amersham Pharmacia Biotech AB.
  • the term "dispensation devices” refer to devices used for dispensation of liquid aliquots to microdevices in which the aliquots are further processed.
  • Microdevices in this context comprise microfluidic devices as well as devices in which no transportation of liquid is taking place, for instance wells that are arrayed in a plate.
  • Typical dispensation devices include drop dispensers and syringe dispensers, and have been described among others by Gyros AB/Amersham Pharmacia Biotech AB.
  • outlet of an outlet microconduit may be tightly connected to an inlet port of a microfluidic device.
  • dispensers utilize transport microconduits in the form of pins and needles (for instance open tubes) in/on which liquid can be retained by capillarity during transport to a target area on a microdevice (US 5,957,167 and 6,116,297 (Pharmacopeia) and WO 0119518 (Aclara), each of which is incorporated herein by reference in its entirety.
  • liquid aliquots refer to an amount of a liquid that is processed in a given protocol and may have the same and/or different volumes and are typically in the ⁇ l-range, for example, but not limiting to volumes in the range ⁇ 1,000 ⁇ l, such as ⁇ 100 ⁇ l or ⁇ 10 ⁇ l and includes nanolitre (nl) or picolitre (pi) volumes, i.e. ⁇ 5,000 nl, e.g. ⁇ 1,000 nl or ⁇ 500 nl or ⁇ 5,000 pi.
  • protocol or "process protocol” is a description of a process and may refer to the order, kind etc of the various steps of the process, such as an experiment.
  • a protocol may include temperature, content of the liquid(s) etc used in the invidual steps, for instance reagents, protocol enhancers etc.
  • a protocol may describe an analytical, preparative, synthetic etc process. Analytical processes are often called assays protocol and then describes an assay.
  • protocol enhancer refers to an exogenous substance that is a non-reactant but can remedy microfluidic protocols or enhance the results of the protocols. This means that the protocol enhancer is not a reactant in the sequence of reactions leading to the product of the process contemplated. Thus a substance/compound that is used for precipitation of disturbing contaminants in an analyte sample is a protocol enhancer even if it is a reactant in the precipitation reaction. Compare that an Ig-preparation according to the 3nd subaspect of the transport embodiment of the invention is used for neutralizing heterophilic antibodies in a sample that contains an analyte.
  • the protocol enhancer is exogenous in the sense that it is separately added to a liquid containing a reactant of a protocol, e.g. an analyte.
  • a reactant e.g. an analyte
  • a protocol enhancer are not originally present together in the same liquid/sample.
  • dissolved contemplates that the dissolved entity is a solute and/or suspended in the liquid.
  • Useful amphophilic macromolecular substances are typically inherently micelle-forming in water at room temperature (25°C). This reflects that a suitable substance should have pronounced surface-active properties by comprising hydrophilic and hydrophobic regions in separate parts and/or ends of the molecule. It is believed that the micelle-forming property as such is not important for the invention but merely reflects basic structural features that are important for accomplishing the objects of the invention.
  • the exogenous amphiphilic macromolecular substance is typically a non- reactant with respect to the assay reactions, i.e. the reactions leading to formation of the product measured.
  • protocol enhancers of the invention is present in a liquid together with a reactant it is preferably in admixture with the reactant before being dispensed to a microdevice, such as a microfluidic device. This in particular applies to analytes.
  • the concentration of the amphiphilic substance can be below or above the critical micelle concentration (at the temperature of use) (cmc).
  • Micelles of the substance thus may or may not be present. Presence of micelles might have a negative impact on the reliability of the results obtained, in particular if micelles are present together with a reactant, such as an analyte.
  • a reactant such as an analyte.
  • micelles of the macromolecular substance are included, for instance with the concentration of the amphiphilic macromolecular substance being above cmc.
  • Appropriate concentrations of the amphiphilic substance can typically be found, in the interval ⁇ 5 %, such as ⁇ 2 % or ⁇ 1% or 0.5 % and/or > 0.001 %, such as 0.005 % or > 0.01 % (w/v-%).
  • the amphiphilic macromolecular substance is thus soluble in the liquid in which it is present and typically has a molecular weight > 3,000 daltons such as > 5,000 daltons or > 10,000 daltons.
  • the molecular weights of natively occurring amphiphilic macromolecular substances are often ⁇ 150,000 daltons, such as ⁇ 100,000 daltons or ⁇ 50,000 daltons.
  • the upper limit for molecular weight can be considerably higher, e.g. 10 7 daltons or 5x10 6 daltons or 10 6 daltons. These ranges apply to unfragmentd forms of the substance.
  • hydrophilic regions of the macromolecular substance there are typically one or a plurality of selected among charged and/or non-charged heteroatom-containing groups where the heteroatom typically is oxygen and/or nitrogen.
  • the heteroatom typically is oxygen and/or nitrogen.
  • there may be one or a plurality of positively or negatively charged groups such as quaternary, tertiary, secondary and primary ammonium groups, benzidium groups, phosphate or phosphonate groups, sulphate or sulphonate groups, carboxy groups etc.
  • Some of these groups have a pH-dependent charge in the pH range typically contemplated (pH 1-12, such as 2-11).
  • Typical uncharged hydrophilic groups are alhoholic hydroxy (primary, secondary or tertiary), amido groups, ester groups, repetitive lower alkylene oxide groups, such as repetitive ethylene oxide groups, etc.
  • Lower alkylene in this context primarily means C 2-4 groups and the term "repetitive" includes that different alkylene oxide groups may be involved.
  • hydrophobic regions of the macromolecular substance there typically are one or a plurality of alkyl and/or aryl groups. Corresponding di- and polyvalent groups/structures may also be present.
  • a hydrophobic alkyl group preferably contains one, two, three, four or more sp 3 -hybridized carbons binding to only hydrogen and carbon and possibly also to fluoro and/or other halogens such as chloro.
  • More specific groups that may be present are selected amongst methyl, ethyl, pro ⁇ -1-yl, prop-2-yl, 2-methyl-prop-l-yl, but-2-yl, phenyl, benzyl, CH 3 -S-CH 2 -, HSCH 2 -, 1 -methyl-prop- 1-yl etc, many of which are common in biopolymers that typically exhibit peptide structure or are derivatized to exhibit such groups.
  • the polymer chain may be branched or straight. The chain is typically not cross-linked. If cross-linked it is only slightly without making the polymer as such insoluble in water and/or other liquids.
  • the advantageous amphiphilic macromolecular substances have a polypeptide structure, with the preferred ones being found amongst amphiphilic milk proteins, such as amongst the caseins with ⁇ -casein being more preferred than others.
  • An amphiphilic macromolecular milk protein may be used together with other milk proteins, such as in defatted milk preparations, possibly in dry form.
  • fragments, aggregates and derivatives of amphiphilic macromolecular substances, typically with micelle-forming properties are included when a parent form is discussed if not otherwise is apparent from the context. It follows that also micelle-forming polymeric substance other than milk proteins, such as other polypeptides or proteins and synthetic polymers are candidates to be used in the invention.
  • Such candidates may have a high degree of homology with ⁇ -casein with respect to the spacing of hydrophobic and hydrophilic monomelic units, such as amino acid residues, if they have polypeptide structure.
  • Examples of potentially useful synthetic polymers are block copolymers comprising hydrophobic regions, for instance with one hydrophobic and one hydrophilic end region, a central hydrophobic region flanked by hydrophilic end regions. This can be illustrated with linear block copolymers, such as the Pluronics that typically have a central polypropyleneoxide region flanked on each end by a polyethyleneoxide region.
  • useful amphiphilic macromolecular protocol enhancers are non-ionic or ionic. Ionic variants may be anionic, cationic, or zwitterionic.
  • an immunoglobulin preparation (Ig, immunoglobulin) may be included as a protocol enhancer. Since immunoglobulins are macromolecular species the Ig preparation may alternatively be coated on the inner surface of transport microconduits and/or elsewhere throughout a microchannel structure.
  • the Ig-preparation maybe of a certain class, such as IgA, IgD, IgE, IgG, and IgM, or subclass.
  • An Ig preparation is included in order to neutralize heterophilic antibodies that may be present together with the analyte in a sample. Compare Kricka L., Clin. Chem. 45 (1999) 942-956.
  • the Ig preparation should thus contain antigenic epitopes that are also present on immunoglobulin from the same species as the species from which an antibody reagent used in the protocol derives. Due to cross-reactivities between species exact species-matching is not required. If used as protocol eenhancers, Ig preparations should be present in excessive amounts compared to the amount of heterophilic antibodies present in a liquid sample. For a liquid aliquot to be transported in the protocol this means that the concentration of Ig preparation typically is within the interval 0.001- 5 % such as 0.01-2 % or 0.01-1 % (w/w-%), preferably in aliquots also containing the analyte.
  • the Ig-preparation may also be present in a liquid aliquot to be transported according to the invention together with a reactant (anti-An) that have affinity for the analyte or some other reactant used.
  • a reactant anti-An
  • concentration ranges given also apply if the aliquot/liquid is a coating solution.
  • immunoglobulin immunoglobulin
  • Ig immunoglobulin
  • Ig-preparation immunoglobulin-preparation
  • Preferred salts can be found amongst soluble salts of group I metals (K, Na etc) with the negative ion typically deriving from a strong acid, i.e. being SO 4 2" , a halide ion, such as F “ , Cl " , etc.
  • the total concentration of salt as defined above should be > 0.10 and preferably within the interval 0.15 - 3 M, such as 0.15 - 2 M or 0.25-1 M, e.g. in a liquid aliquot containing a reactant, such as an analyte.
  • Appropriate salts should be inert towards the various reactants used and should not create disturbing precipitations.
  • Salt as referred to in this paragraph includes that salt has been added in excess of the salt, if any, that derives from an original liquid sample containing a reactant of the protocol concerned.
  • Surfactants are examples of additional protocol enhancers that may be used in combination with enhancers according to one or more of the 1 st to 3 rd subaspects. Suitable surfactants are typically detergents and have a moderate to low molecular weight, such as ⁇ 50,000 daltons, such as ⁇ 20,000 daltons or ⁇ 10,000 daltons or ⁇ 5,000 daltons or ⁇ 3,000 daltons or ⁇ 2,000 daltons.
  • Non-ionic and ionic surfactants where the ionic ones are anionic, cationic or zwitterionic. They may be synthetic (tensides) or native. They are typically micelle-forming in the same manner as the macromolecular substance. They exhibit a hydrophobic group and a hydrophilic group selected amongst the same hydrophilic and hydrophobic groups that may be present in the amphiphilic macromolecular substance or in the various kinds of reactants discussed in this specification.
  • Appropriate concentrations of the surfactant to be used in combination with one or more of the l st -3rd subaspects of the invention in the aliquot to be transported are typically selected within 0.001-5 %, such as 0.01-3 % or 0.05- 2 % (w/v) or 0.01-1 % (w/v). These concentration ranges apply to detergents and tensides, for instance, and other additional protocol enhancers that are originally present together with a reactant, such as an analyte, e.g. in a biological fluid used for obtaining a liquid aliquot to be used in the invention.
  • the above-mentioned protocol enhancers may be combined with still other substances that are known to reduce the undesired interactions discussed above.
  • These other substances/enhancers may be present in the same liquid aliquots as those that contain an enhancer of the 1 st - 3 rd subaspects. Alternatively they may be introduced in one or more separate aliquots that subsequently may or may not be mixed with an aliquot containing an enhancer of l st -3 rd subaspects.
  • the concentration of this kind of other substance/enhancer in a liquid aliquot to be transported in accordance with the invention is typically in the interval 0.001- 5 %, such 0.01-3 % (w/v).
  • a typical such other substance/enhancer is separately added serum albumin.
  • a hydrophobic group typically exhibits an aromatic group and/or a cyclic, branched or straight alkyl or alkylene group.
  • Aromatic groups may be illustrated with phenyl and benzyl groups in substituted or unsubstituted forms.
  • Alkyl or alkylene groups may comprise hydrocarbon chains containing one, two, three or more sp 3 -hybridised carbon atoms, such as in methyl, ethyl, prop-1-yl, prop-2-yl, 2-methyl-prop-l-yl, but-2-yl, phenyl, benzyl, CH 3 - S-CH 2 -, HSCH 2 -, 1 -methyl-prop- 1-yl etc.
  • carbon chains in bioorganic molecules have less than 30 carbon atoms.
  • Charged groups are positively and/or negatively charged and include that a reactant has both kinds of groups and a net positive charge, a net negative charge, or a net zero charge.
  • the net charge of the reactant or of a group may be pH-dependent, e.g. the reactant may be zwitterionic, such as an ampholyte with an isoelectric point (pi).
  • the reactant may be zwitterionic, such as an ampholyte with an isoelectric point (pi).
  • pi isoelectric point
  • These kinds of groups/reactants may potentially interact with inner surfaces that expose hydrophobic groups or groups that have the opposite charge compared to a charged group on a reactant. Interaction may also occur if the charged group of the inner surface is present within a layer below an inner surface, for instance within deficient coating layers, such as in so-called pin-holes, or within a surface layer of the material in which the microchannel structure has been fabricated.
  • the reactants concerned may be illustrated with analytes, reagents/reactants exhibiting a detectable group, reagents/reactants exhibiting an immobilizing group or tag, etc and are typically bioorganic compounds.
  • Reactants that are bioorganic compounds and exhibit hydrophobic groups may be found amongst proteins and polypeptides, glycolipids and lipoproteins, lipids such as many steroids, fatty acids and fatty acid derivatives such as esters including glycerides, and higher bio-alcohols (with five or more carbon atoms) and their derivatives.
  • Reactants that are bioorganic compounds and/or exhibit charged groups are primarily found amongst proteins including other substances exhibiting peptide structure, and nucleic acids including other substances exhibiting nucleotide structure.
  • a bioorganic reactant, that contains a polypeptide structure typically also contains a) one or more positively charged or chargeable groups, c) one or more negatively charged or chargeable groups and/or b) one or more hydrophobic groups. These kinds of groups are represented by the presence of the following amino acid residues:
  • hydrophobic amino acids are: alanine (1.8), cysteine (1.5), leucine (3.8), isoleucine (4.5), methionine (1.9), phenylalanine (2.8) and valine (4.2).
  • amfolyte reactan ⁇ that has a pi that is larger than the pH-value of the aliquot in which the reactant is transported may benefit more than an amfolyte reactant 2 for which pi is less than this pH.
  • This trend is likely to also be at hand at least partially if amfolyte reactanti has a pi that is larger than pH minus 1 where pH is the pH-value of the liquid aliquot.
  • This trend is based on the behavior of surfaces exposing negatively charged groups, such as plastic (Vplasma hydrophilized surface coated with non-ionic hydrophilic polymer by electrostatic interaction.
  • a positively charged or a more acidified form of a pH-dependent reactant may thus benefit more than a less acidified form, e.g. a basic polypeptide analyte may benefit more than an acidic polypeptide analyte. It is not unlikely that further investigations will show that benefits may also be accomplished for negatively charged reactants, for instance zwitterionic reactants, such as amfolyte reactants that have pi ⁇ pH + 1, such as ⁇ pH, of the aliquot in which it is transported, in particular if microconduits that have inner surfaces comprising positively charged groups are used.
  • zwitterionic reactants such as amfolyte reactants that have pi ⁇ pH + 1, such as ⁇ pH
  • a reactant that has a mean hydropathy index > - 0.5, such as > - 0.1 > 0 or > 0.1 or > 0.2 or > 0.3 or > 0.4. Due to the complexity of polypeptide/protein structure it may many times be better to apply these mean hydropathy ranges to one or more parts of the sequence of the polypeptide structure of a reactant to find reactants that may benefit from the invention.
  • a part sequence comprises from 2, 3, 4, 5, 6 and more consecutive amino acid residues and/or between 1-99 %, such as 2-99% or 4-50 % or 4-25 % or 4-10 % or 1-6 % of the total number of amino acid residues in the Ml length sequence.
  • Reactants comprising 1, 2, 3, 4, 5, 6, 7 or more consecutive hydrophobic amino acid residues typically represent reactants that may benefit from one or more of the subaspects of the instant invention.
  • microconduits are primarily present in: a) dispensation devices, and b) microchannel structures of microfluidic devices.
  • a transport microconduit typically comprises a dispensation orifice and thus primarily comprises the outlet part of the liquid transportation system of a dispensation device, i.e. the part from which a liquid leaves the device for a target area of a microdevice.
  • the dispensation device may be a drop-dispenser, plunge driven dispenser (e.g. syringe) or based on other pump mechanisms as is well known in the field.
  • Other kinds of dispensers use transport microconduits in the form of pins or needles to which the liquid aliquot is retained by surface forces, e.g. capillary forces, during the transport.
  • the term "transport microconduit" in the context of dispensation is generic and refers more to transport to a microdevice than transport through a microconduit.
  • the microdevice may be a plate containing a number of wells (microtitre plate), a microfluidic device comprising one or more microchannel structures, etc. The liquid transferred to the microdevice is further processed in the wells, microchannel structures etc.
  • dispensers in which a larger liquid aliquot is first collected in the transport microconduit and then dispensed in minor aliquots in sequence to different target areas of one or more microdevices.
  • the transport microconduits of dispensation devices may be fabricated in glass, metal, plastics, ceramics etc.
  • the inner surfaces may be pre-treated as is well-known in the field for liquid transportation systems, i.e. made wettable/hydrophilic and/or equipped with a surface with lowered fouling activity.
  • the term "transport microconduit" in a microfluidic device/microchannel structure contemplates all parts of the microchannel structure through which liquid shall pass except those cavities/microconduits in which heterogeneous reactions of a protocol are to take place.
  • the term includes also waste chambers/reservoirs (if present).
  • a heterogeneous reaction is a reaction between a dissolved reactant and a solid phase bound reactant.
  • transport microconduit in the context of the invention shall refer to meaning 1 if not otherwise being clear from the context.
  • reaction microcavities/microconduits in which other kinds of reactions (non-heterogeneous reactions) are excluded from being transport microconduits.
  • Typical such other reactions are homogeneous reactions of the protocol, i.e. reactions between reactants in dissolved form.
  • transport microconduits are microconduits that are only used for transport of liquid aliquots that may or may not contain reactants.
  • a "transport microconduit" in the third meaning has the sole function of transporting liquid between functionalities, such as inlet openings, volume-definition units, valves, vents, mixing units, liquid routing units, units for particle separation from liquids, units for removal of contaminants and disturbing substances, reaction units, detection units etc.
  • a microfluidic device comprises one, two or more microchannel structures each of which is intended for carrying out a desired protocol by transporting and processing one or more aliquots of liquid.
  • the number of microchannel structures/device is typically > 10, e.g. > 25 or > 90 or > 180 or > 270 or > 360.
  • Each of the microchannel structures contains 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 (depth and/or width).
  • the nl-range has an upper limit of 5,000 nl. In most cases it relates to volumes ⁇ 1,000 nl, such as ⁇ 500 nl or ⁇ 100 nl.
  • Each of the microchannel structures typically comprises one, two, three or more functional parts selected among: (a) inlet arrangements comprising for instance an inlet port/inlet opening, possibly together with a.
  • volume-metering unit metering/defining of liquid volumes to be processed
  • microconduits for liquid transport meaning 3 above
  • reaction microcavities d) mixing microcavities
  • f) units for separating dissolved or suspended components in the sample from each other for instance by capillary electrophoresis, chromatography and the like
  • detection microcavities h
  • waste conduits/ microcavities i) valves; j) vents to ambient atmosphere; etc.
  • a functional part may have one or more functionalities, e.g. a reaction microcavity and a detection microcavity may coincide.
  • 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 the disc plane, where n is an integer > 2, 3, 4 or 5, preferably oo (C 00 ).
  • the disc may be rectangular, such as in the form of a square, or have other polygonal forms. It may also be circular (C 00 ).
  • centrifugal force may be used for driving liquid flow, e.g. by spinning the device around a spin axis that typically is perpendicular or parallel to the disc plane. In the most obvious variants at the priority date, the spin axis coincides with the above- mentioned axis of symmetry.
  • each microchannel structure comprises an upstream section that is at a shorter radial distance than a downstream section relative to the spin axis.
  • the preferred devices are typically disc-shaped with sizes and forms similar to the conventional CD-format, e.g. sizes that corresponds CD-radii that are the interval 10% - 300 % of the conventional CD-radii.
  • Microchannels/microcavities of a microfluidic device 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 See WO 9116966 (Pharmacia Biotech AB), WO 0154810 (Gyros AB), and WO 03055790 (Gyros AB).
  • the material of the substrates may be selected among various kinds of inorganic and organic material, for instance polymeric material, such as plastics.
  • an essential part of the inner surfaces of the microchannel structures should have water contact angles ⁇ 90°, such as ⁇ 60° or ⁇ 40° or ⁇ 30° or ⁇ 20° at the temperature of use or 25°C. At least two or three of the inner walls enclosing the channels should comply with this range. Surfaces in passive valves, anti-wicking means etc are excluded from these general rales. Surfaces made in plastics typically need to be hydrophilized.
  • Parts of a microchannel structure that are used for liquid transportation or otherwise intended to be in contact with an aqueous liquid are preferably hydrophilic.
  • the wettability of inner surfaces of such a part supports filling the part with liquid by capillarity (self suction) provided a front of the liquid has passed the entrance of the part, e.g. passed a valve function at the entrance.
  • Non-wettable surface breaks may be introduced at predetermined positions in the inner walls of the microchannel structures before covering the uncovered microchannel structures (WO 9958245, Amersham Pharmacia Biotech AB) and WO 0185602, Amic AB & Gyros AB, each of which is incorporated herein by reference). For aqueous liquids this means hydrophobic surface breaks. Surface breaks may be used for controlling the liquid flow within the structures, e.g. in anti-wicking, passive valves, directing liquids etc.
  • a microchannel structure comprising at least one fluidic function based on surface tension such as in capillary valves, anti-wicking means, liquid- directing etc might be simpler to deal with if the macromolecular protocol enhancer is present in dissolved form than in pre-coated form, e.g. the amphiphilic macromolecular substance and/or the Ig- ⁇ reparation.
  • the macromolecular protocol enhancer is present in dissolved form than in pre-coated form, e.g. the amphiphilic macromolecular substance and/or the Ig- ⁇ reparation.
  • This concern in particular applies to structures in which these functionalities are based on hydrophobic/non-wettable surface breaks in otherwise hydrophilic microconduits.
  • a reaction microcavity may comprise a solid phase typically exposing a solid phase bound reactant, such as a reactant that is used in steps leading to formation of the final product of the protocol.
  • the solid phase may expose a reactant for removing contaminants and/or disturbing substances that are present in a liquid aliquot passing the solid phase/reaction microcavity.
  • the solid phase may be the inner walls of the reaction microcavity or a porous bed retained in the reaction microcavity.
  • a porous bed is typically a) a porous monolith, or b) a population of porous or non-porous particles that are packed to a porous bed.
  • the material in the porous bed e.g. the particles
  • the particles and other forms of solid phases are typically hydrophilic in the case the liquid flow is aqueous.
  • hydrophilic encompasses that a porous solid phase, e.g. a packed bead, will be penetrated by water by self- suction.
  • the term also indicates that the surfaces of the particles shall expose a plurality of polar functional groups in which there is a heteroatom selected amongst oxygen, sulphur, and nitrogen.
  • a hydrophobic particle or porous monolith may be hydrophilized, for instance by introducing hydrophilic groups.
  • the coating and hydrophilization technique may be similar to the technique presented in WO 9529203 (Pharmacia Biotech AB), WO 9800709 (Pharmacia Biotech AB, Arvidsson & Ekstr ⁇ m), WO 0146637 (Gyros AB), WO 0056808 (Gyros AB) and WO 03086960 (Gyros AB), each of which is incorporated herein by reference.
  • the techniques for immobilization of an reactant to a solid phase may be selected amongst those that are commonly known in the field. Immobilization may thus be via covalent bonds, affinity bonds, physical adsorption (mainly hydrophobic interaction) etc.
  • biospecific affinity bonds that can be used are bonds a) between streptavidin and a biotinylated affinity reactant, b) between high affinity antibody and a haptenylated affinity reactant etc, and vice versa.
  • the liquid transport is typically part of a protocol in which one or more aliquots of liquid are processed within a microdevice, such as a microfluidic device. At least one of the aliquots contains a reactant of the protocol.
  • the protocol may be analytical, preparative, synthetic etc. In principle any kind of synthetic or preparative protocol carried out in a microdevice means that the results are analyzed to find out something about the actual reactions carried out, about the reactants involved, in other words most preparative and synthetic protocols are part of an analytical protocol.
  • analytical protocols also comprises to study how various reaction conditions/variables such as concentration of a reactant, pH, ionic strength, presence of detergents, type of salts, temperature, presence of various components etc interfere with the result of a reaction protocol. This latter kind of analytical protocols typically comprises comparative studies in which one or more reaction conditions are changed. For these protocols the term "analyte" means the variable(s) that is(are) changed between the comparative experiments.
  • the typical protocols are applicable to biological science and/or chemistry including medicine, biochemistry, molecular biology, environmental science, diagnostics, inorganic, organic and/or analytical etc chemistry etc.
  • a typical protocol utilizes a bioorganic reactant, e.g. the analyte, while one or more of the other reactants may be inorganic, such as a metal ion or an inorganic anion.
  • synthetic organic compounds mimicking a bioorganic compound/reactant are also bioorganic.
  • analytes are a) hormones, such as peptide hormones and steroid hormones, b) hormone receptors, c) growth factors, d) components of enzymatic systems, such as enzymes, enzyme substrates, cofactors, coenzymes, cosubstrates etc, e) immune modulators such as cytokines, chemokines including interferons and interleukins, f) inflammatory mediators, such as ECP, MPO etc, g) antigens and haptens, h) drugs, i) immunoglobulins such as immunoglobulin as such and its subclasses, j) blood clotting factors, k) complement factors, 1) tissue, m) microorganisms such as bacteria, mould, fungi, viruses, prions, etc, n) occupational health molecular indicators, o) toxins, p) nucleic acids and other compounds exhibiting nucleotide structure, etc.
  • hormones such as peptide hormones and steroid hormone
  • Typical protocols are ligand-receptor assays, catalytic assays, cell- based assays etc. Included are also sample preparation for analyses outside the microdevice, for instance by MS (US 6,812457 and US 6,812,456 (Gyros AB)).
  • This kind of protocols comprises a step in which an affinity complex comprising the analyte and an affinity counterpart to the analyte (anti-An) is formed.
  • an affinity complex comprising the analyte and an affinity counterpart to the analyte (anti-An) is formed.
  • the amount of (a) complex formed, and/or (b) uncomplexed analyte, and/or (c) uncomplexed anti-An is/are correlated with the starting amount of analyte, e.g. the concentration/amount/activity of the analyte in a fluid from which the analyte derives, e.g. a biological fluid.
  • the measurement of (a), (b) or (c) is often facilitated by the use of an affinity reactant, e.g.
  • Anti-An or analyte analogue which comprises a detectable group (detectable reactant), such as a label, and is capable of forming an affinity complex that contains the appropriate one of entities (a)-(c).
  • detectable reactant such as a label
  • antibody includes intact native antibodies as well as antigen-binding fragments and derivatives thereof, such as Fab, F(ab) 2 , single chain antibodies, chimeric antibodies, conjugates containing one or more antibody moieties, synthetic forms trying to mimicking the antigen specificity of an antibody.
  • Antibodies may be chemically and/or recombinantly produced.
  • Receptor-ligand assays can be divided in two main groups 1) heterogeneous assays and 2) homogeneous assays.
  • a heterogeneous assay comprises a separation step before the measurement is talcing place, i.e. the affinity complex is separated from the analyte and/or from anti-An not incorporated into the complex, or the affinity complex containing the detectable reactant is separated from the portion of detectable reactant that is not incorporated into this complex.
  • Separation is facilitated by the use of an immobilized or immobilizable form of anti-An or of an analogue to the analyte (An-analogue). In a homogeneous assay this kind of separation step is not included. In stead one makes use of an affinity reactant that exhibits a detectable group for which the feature to be measured changes as a consequence of affinity complex formation.
  • the inventive concept can be applied to both heterogeneous and homogeneous receptor-ligand assays.
  • Receptor-ligand assays can also be divided into: 1) competitive assays, and 2) non-competitive assays.
  • an analyte analogue (An-analogue) is allowed to compete with or inhibit the binding of an analyte to anti-An.
  • Anti-An is typically present in limiting amounts.
  • Competitive assays also include a) so-called displacement assays which typically comprises a first step in which a limiting amount of anti-An is saturated with An- analogue whereafter the analyte is allowed to displace complex-bound An-analogue, and b) back-titration methods in which an excess of anti-An is reacted with An whereafter residual An binding sites is reacted with An-analogue.
  • Competitive assays may be homogeneous or heterogeneous.
  • Typical analytes that are used in competitive assays are haptens or antigens.
  • the analytes are typically relatively small, e.g. ⁇ 10,000 daltons, such as ⁇ 5,000 daltons or ⁇ 2,000 daltons.
  • Typical anti-Ans are antigen specific antibodies.
  • Typical analytes are haptens or antigens.
  • Sandwich assays are one of the most popular.
  • an analyte is tethered between two reactants which each is an affinity counterpart to the analyte (ahti-An).
  • Typical analytes are relatively large with molecular weights > 2,000 daltons, such as > 5,000 daltons > 10,000 daltons, and are antigens including also immunoglobulins and antigen-specific antibodies.
  • Typical anti-Ans are anti-An antibodies and other affinity reactants that are affinity counterparts to the analyte at issue.
  • the analyte is an antigen specific antibody
  • one or both of the anti-AnS' may be an antigen analogue while the remaining anti-An, if any, is an anti-An antibody directed towards the Fc part of the analyte or some other affinity reactant that has affinity for a non-antigen binding of the antibody.
  • this means that one of the anti-Ans is in immobilized or immobilizable form while the other exhibits the detectable group.
  • Other kinds of non-competitive assays utilize only one anti-An that exhibits a detectable group for which the feature to me measured changes when the reactant becomes incorporated into an affinity complex together with the analyte.
  • Well-known examples are scintillation proximity assays (SPA).
  • Ih catalytic assays the analyte (An) is a component of a catalytic system.
  • catalytic system primarily contemplates biocatalytic systems, for instance enzymatic systems that are based on enzymatically active proteins or synthetic variants thereof.
  • catalytic system also encompasses so called coupled catalytic systems in which a single catalytic system is combined with other catalytic system and/or other reaction systems such as an antigen-antibody reaction system.
  • protocol enhancers which are not substrates for the catalytic system to be studied. Casein and other enhancers having polypeptide structure should be avoided if the catalytic system at issue has proteolytic activity. Synthetic amphiphilic macromolecular substances or inert protein substrates as protocol enhancers may then be more appropriate choices.
  • Enzyme systems may be selected amongst: 1) Oxidoreductases
  • a component of catalytic system may be native or may have been produced synthetically or recombinantly.
  • the component may exhibit amino acid structure, peptide structure, such as oligo- or polypeptide structure, nucleotide structure, such as oligo- or polynucleotide structure, carbohydrate structure such as oligo- or polypeptide structure, lipid structure, steroid structure, hormone structure etc, i.e. structures that may be present in reactants of other protocols to which the invention is applicable. Synthetic compounds, for instance deriving from combinatorial libraries, potentially mimicking native variants of components of catalytic systems are included.
  • Catalytic assays to which the present invention is applied may be homogeneous or heterogeneous.
  • a homogeneous catalytic assay contemplates that all components of the system used are in dissolved form, except for the product that may be either in dissolved or in insoluble or immobilized form.
  • a heterogeneous catalytic assay accordingly contemplates that one or more of the components other than the product formed are in insoluble or immobilized form.
  • Catalytic assays typically utilize detectable groups on the substrate and/or the product.
  • the detectable groups may typically be selected amongst the same alternatives as for receptor-ligand assays.
  • Cell based assays means that one of the reactants transported within the transport microconduit is a suspension of cells.
  • the cells typically are viable and subjected to one or more agents whereupon the effect of the agents on the cells are studied, possibly by utilizing a receptor-ligand protocol or a catalytic protoc ol of the type well-known in the field, for instance as discussed above, or by studying cell morphology, motility, proliferation etc. See also US 6,632,656, which is incorporated herein by reference in its entirety.
  • the agent concerned may be of any kind including also phages, viruses and the like, temperature, time, humidity, gas pressure such as CO 2 and/or O 2 pressure, nutrients, toxins, vitamins, hormones etc.
  • the cells may be grown outside or inside the device in the presence or absence of the agent(s) concerned before the effects are studied. After subjecting the cells to the agent(s) the cells may be killed inside the microdevice, for instance lysed or fixated, while being retained in the microdevice where various released or bound cell components then are investigated.
  • Typical cells may be nucleated or not nucleated, anchorage-dependent or non-anchorage dependent, primary or secondary, bacterial or non-bacterial, plant cells or animal cells etc.
  • ATCC American Type Culture Collection
  • Cell-based assays according to the invention also may include studying of living tissue within a microdevice.
  • the tissue may comprise a cell.
  • the tissue may be part or separated from an organism.
  • the cell or tissue may be comprised in at least one organism.
  • the organism may be, but is not limited to, an eubacteria, an archaea, an eukaryote or a virus.
  • the following are examples of cells that may be studied using the methods and compositions of the present invention.
  • the organism is an eubacteria.
  • the eubacteria may be, but is not limited to, an aquifecales; a thermotogales; a thermodesulfobacterium; a member of the thermus deinococcus group; a chloroflecales; a cyanobacteria; a firmicutes; a member of the leptospirillum group; a synergistes; a member of the chlorobium flavobacteria group; a member of the chlamydia verrucomicrobia group, including but not limited to a verrucomicrobia or a chlamydia; a planctomycetales; a flexistipes; a member of the fibrobacter group; a spirochetes; a proteobacteria, including but not limited to an alpha proteobacteria, a beta proteobacteria, a delta
  • the organism is an archaea (a.k.a. archaebacteria; e.g., a methanogens, a halophiles, a sulfolobus).
  • the archaea may be, but is not limited to, a korarchaeota; a crenarchaeota, including but not limited to, a thermofilum, a pyrobaculum, a thermoproteus, a sulfolobus, a metallosphaera, an acidianus, a thermodiscus, a igneococcus, a thermosphaera, a desulfurococcus, a staphylothermus, a pyrolobus, a hyperthermus or a pyrodictium; or an euryarchaeota, including but not limited to a halobacteriales, methanomicrobiales, a methanobacterial
  • the organism is an eukaryote (e.g., a protist, a plant, a fungi, an animal).
  • the eukaryote maybe, but is not limited to, a microsporidia, a diplomonad, an oxymonad, a retortamonad, a parabasalid, a pelobiont, an entamoebae or a mitochondrial eukaryote (e.g., an animal, a plant, a fungi, a stramenopiles).
  • the mitochondrial eukaryote may be, but is not limited to, a metazoa (e.g., an animal), a myxozoa, a choanoflagellate, a fungi (e.g., a mushroom, a mold, a yeast, a chytrid), a green plant (e.g., a green algae, a land plant), a cryptomonad, an ancyromona, plasmodiophorid, a rhodophyta, a centrohelid heliozoa, a cyanophorid, an alveolate (e.g., a dinoflagellate, a sporozoan, a ciliate), a stramenopile (e.g., a brown algae, a diatoms, an oomycete, a chrysophyte), an acantharea, a vampyrellid, a metazoa (e.g.,
  • the eukaryote is a metazoa (e.g., an animal).
  • the metazoa may be, but is not limited to, a porifera (e.g., a sponge), a cnidaria (e.g., a jellyfish, an anemone, a coral), a ctenophora (e.g., a comb jelly), an arthropoda (e.g., an insect, a spider, a crab), an annelida (e.g., a segmented worm), a pogonophora, a vestimentifera, an echiura, a mollusca (e.g., a snail, a clam, a squid), a sipuncula, a nemertea (e.g., a ribbon worm), a platyhelminthes (e.g., a flatworm), a chordata (e
  • a porifera e
  • the vertebrate may be a terrestrial vertebrate (e.g., a frog, a salamander, a caecilian, a reptile, a mammal, a bird) or a non terrestrial vertebrate (e.g., a sharks, a ray, a sawfish, a chimera, a ray finned fish, a lobe finned fish).
  • a terrestrial vertebrate e.g., a frog, a salamander, a caecilian, a reptile, a mammal, a bird
  • a non terrestrial vertebrate e.g., a sharks, a ray, a sawfish, a chimera, a ray finned fish, a lobe finned fish.
  • the mammal may be a monotremata (e.g., a platypus, an echidna), a multituberculata, a marsupialia (e.g., an opossum, a kangaroo), a palaeoryctoids or an eutheria (e.g., a placental mammal).
  • a monotremata e.g., a platypus, an echidna
  • a multituberculata e.g., a marsupialia (e.g., an opossum, a kangaroo), a palaeoryctoids or an eutheria (e.g., a placental mammal).
  • the eutheria may be, but is not limited to, an edentata (e.g., an anteater, a sloth, an armadillo), a pholidota (e.g., a pangolin), a lagomorpha (e.g., a rabbits), a glires, a rodentia (e.g., a mouse, a rat, a squirrel, a gopher, a porcupine, a beaver), a macroscelidea (e.g., an elephant shrew), a primates (e.g., a monkey, a lemur, a gorilla, a chimp, a human), a scandentia (e.g., a tree shrew), a chiroptera (e.g., a bat), a dermoptera (e.g., a colugo, a flying lemur), an insecti
  • an edentata
  • eukaryote is a fungi.
  • a fungi may be, but is not limited to, a chytridiomycota (e.g., a water mold, an allomyces), a zygomycota (e.g., a bread mold, a rhizopus, a mucor), a basidiomycota (e.g., a mushroom, a rust, a smut) or an ascomycota (e.g., a sac fungi, a yeast, apenicillium).
  • a chytridiomycota e.g., a water mold, an allomyces
  • zygomycota e.g., a bread mold, a rhizopus, a mucor
  • basidiomycota e.g., a mushroom, a rust, a smut
  • an ascomycota e.g., a sac fungi,
  • the eukaryote is a green plant.
  • a green plant may be, but is not limited to, a prasinophytes, a chlorophyceae, a trebouxiophyceae, a ulvophyceae, a chlorokybales, a klebsormidiales, a zygnematales, a streptophyta, a charales, a coleochaetales or an embryophytes (e.g., a land plant).
  • the embryophytes may be, but is not limited to, a marchantiomorpha (e.g., a liverwort), an Anthoceromorpha (e.g., a hornwort), a bryopsida (e.g., a moss), a lycopsida (e.g., a lycophyte), an equisetopsida (e.g., a horsetail, a sphenophyte), a filicopsida (e.g., a fern), a spermatopsida (e.g., a seed plant: a flowering plant, a conifer).
  • a marchantiomorpha e.g., a liverwort
  • an Anthoceromorpha e.g., a hornwort
  • a bryopsida e.g., a moss
  • a lycopsida e.g., a
  • the spermatopsida may be, but is not limited to an angiosperm.
  • An angiosperm may include, but is not limited to, a ceratophyllaceae, a nymphaeales, a piperales, an aristolochiales, a monocotyledons, an eudicots, a laurales, a chloranthaceae, a winterales or a magnoliales.
  • the organism may be a virus.
  • the virus may be, but is not limited to, a DNA Virus, including but not limited to a ssDNA virus or a dsDNA virus; a DNA RNA rev transcribing virus; a RNA virus, including but not limited to a dsRNA virus, including but not limited to a -ve stranded ssRNA or a +ve stranded ssRNA; or an unassigned virus.
  • the detectable group is either natively present in a detectable reactant or has been introduced by man as a label onto a reactant.
  • the detectable group is part of the native structure of a reactant, such as a protein, a polysaccharide, a nucleic acid etc. Typical examples are: class, subclass and species specific (IgFc) determinants in immunoglobulins/antibodies.
  • a reactant such as a protein, a polysaccharide, a nucleic acid etc.
  • Typical examples are: class, subclass and species specific (IgFc) determinants in immunoglobulins/antibodies.
  • Conjugates may be obtained by recombinant techniques and/or synthetically by using organic synthetic coupling chemistry.
  • Affinity detectable groups require an extra reactant that comprises an affinity counterpart to the detectable group and a second detectable group. This extra reactant is typically in the form of a man-designed conjugate.
  • Typical affinity detectable groups are members of affinity pairs such as biotin and biotin-binding compounds such as anti-biotin antibodies, avidin, streptavidin, neutravidin etc, complementary nucleic acids, haptens and anti- hapten antibodies, lectins and carbohydrates, IgFc determinants and IgFc binding polypeptides etc.
  • Typical signal-generating groups are particles, metals, chromophors, fluorophors (including fluorogens), radioactive groups, luminophors (including chemi- and bio luminophors), catalytically active groups such as catalysts, substrate, co-substrate, cocatalysts etc.
  • catalytically active groups such as catalysts, substrate, co-substrate, cocatalysts etc.
  • the most popular catalytically active groups are the enzymatically active groups such as enzymes, coenzymes, cofactors, substrates, co-substrates etc.
  • PBS-Tween 15 ⁇ l Tween 20 10% + 14.985 ml PBS (Ix). Diluent for capture antibody and liquid for column wash in microfluidic device (CD)
  • PBS-BSA 1 ml BSA 10% + 9 ml PBS (Ix). Diluent for detection Ab.
  • Samples 13 human serum samples with insulin levels from 9-209 ⁇ U/ml according to RIA.
  • Insulin sandwich assays and heterophilic antibody sandwhich assays as outlined for sandwich in WO 04083108 and WO 04083109 Diluted samples and standards were dispensed to the microfluidic device.
  • heterophilic antibodies were 1-4 ⁇ U/ml, i.e. below the mean value for healthy persons. Measurable neutralization of heterophilic antibodies took place in one of the samples.
  • Serum milieu Protocol enhancers 100 pM in analyte.
  • Amphiphilic macromolecular substance ⁇ -casein, Pluronic F127 and F68 (BASF) (triblock copolymer (EO) m (PO) n (EO) m where EO is ethylene oxide and PO propylene oxide.
  • BASF Pluronic F127 and F68
  • Non-ionic Tween 20 and 80 (Merck), Triton XlOO (Sigma), Pluronic F127 and F68 (BASF), Glucopone (Fluka), Brij 35 (Aldrich); b) Zwitterionic CHAPS (Sigma), Zittergent (Calbiochem), DPC (dodecylphosphocholine); c) Cationic: CTAB (Sigma).
  • Evaluation criteria CV and detection level.

Abstract

L'invention concerne un procédé permettant de transporter dans un conduit de transport microfluidique une aliquote d'un liquide dans lequel est présent un réactif d'un protocole expérimental. Le procédé de l'invention est caractérisé en ce que le réactif est co-transporté avec une substance macromoléculaire amphiphile, et/ou en ce que les surfaces intérieures dudit microconduit sont revêtues par une substance macromoléculaire.
PCT/SE2006/000070 2005-01-17 2006-01-17 Procede permettant de co-transporter un reactif avec une substance macromoleculaire amphiphile dans un conduit de transport microfluidique WO2006075964A1 (fr)

Priority Applications (2)

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JP2007551224A JP2008527371A (ja) 2005-01-17 2006-01-17 微小流体輸送導管の中で両親媒性高分子物質と一緒に反応物を共輸送するための方法
EP06700622A EP1848996A4 (fr) 2005-01-17 2006-01-17 Procede permettant de co-transporter un reactif avec une substance macromoleculaire amphiphile dans un conduit de transport microfluidique

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