Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0605—Metering of fluids
• • 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
  • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• • 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
  • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
• • 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
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
• • 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
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0688—Valves, specific forms thereof surface tension valves, capillary stop, capillary break

Definitions

• the present invention relates to a microfluidic method for determining the amount of an analyte in a liquid sample by the use of a reactant (Re) that is capable of binding to the analyte (An) by affinity, and to a microfluidic device in which the method can be carried out.
• the method is either a competitive receptor-ligand assay, e.g. a competitive immunoassay, or a catalytic assay, e.g. an enzymatic assay.
• Figures 1 gives a generalized flow scheme of a microchannel structure that can be used in the present invention.
• Figures 2a-c illustrate a preferred microchannel structure.
• Figure 2c is the same as figure
• Figure 3 illustrates a set of microchannel structures that has been used in the competitive immunoassays described in the experimental part.
• Figure 4 gives the result of experiment 1 (quantitative immunoassay of substance P).
• Figure 5 gives the result of experiment 2 (quantitative immunoassay of substance NPY).
• Figure 6 illustrates schematically the methodology used in experiment 2
• Microfluidic devices are well known in the field. Asingle device typically comprises a plurality of microchannel structures. The flow scheme of a typical microchannel structure (100) is illustrated in figure 1 and comprises in the downstream direction:
• C a measuring zone (MZ) (103), and (D) an outlet and waste arrangement (OWA) (104).
• MZ measuring zone
• OWA outlet and waste arrangement
• ISA (101) typically contains one, two or more inlet units (IU) (107-111) and may optionally also contain one or more reactant or sample transformation units (RTU) (112-116).
• IU inlet units
• RTU reactant or sample transformation units
• the individual IUs and RTUs may be associated with the same part or with separate parts of a microchannel structure. Further details of microfluidic devices/microchannel structures are discussed under the heading "Micofiuidic devices”.
• the reactant (Re) is an affinity counterpart (anti-An) to both the analyte (An) and to an analogue to the analyte (An-analogue).
• the product P comprises the affinity complex Re — An- analogue in which Re and An-analogue bind directly to each other.
• Catalyst based assays i.e.
• assays that utilize a catalytic system that converts a substrate S to the product P via a transient affinity complex that comprises substrate S (- Re) and one or more other components of the catalytic system.
• substrate S (- Re) substrate S
• One of these other components is the analyte.
• the analyte and substrate S (Re) bind directly (Re — analyte) or indirectly (Re — B — analyte) to each other.
• B is then an affinity counterpart to both the analyte and Re (anti- An 5 Re) and may contain/consist of one or more affinity reactants that also are components of the catalytic system.
• the protocols (a) and (b) when applied to microfluidic devices comprises the steps of: (i) providing a product P that has been obtained according to (a) or (b) above in immobilized form within the measuring zone MZ (103) of the microchannel structure (100) of a microfluidic device,
• step (ii) measuring the amount of product P in the measuring zone MZ (103).
• the conditions for obtaining the product P have been selected such that the amount of product P correlates with the amount of analyte in the sample.
• the correlation means that the amount of analyte in the sample can be calculated (step (iii)) from the measured value for product P obtained in step (ii). Calculation can be made by comparing a measured value with the corresponding value(s) for known amounts (standards, standard curves etc), for instance.
• Conditions include proper selection of reagents including their relative amounts, pH, ion strength etc.
• kinase assay protocols i.e. a particular kind of protocol (b) above.
• Kinases are enzymes that catalyse the transfer of a phosphate group from ATP to a substrate. Their assays are based on the quantification of phosphorylated product or the depletion of ATP. The phosphorylation of substrate occurs at serine, threonine or tyrosine in a specific manner depending on the kinase. Most current kinase assay methods require antibodies, radioactive labeling or indirect measurement of secondary reactions to measure transfer of the phosphate group. For an overview of marketed kinase assays see "How to choose an in vitro kinase assay" (Drug Discovery and development, March 2004, 59-64).
• Protein kinases have key roles in a large number of immune-related diseases, such as cancer, immune diseases, diabetes etc. This has led to extensive efforts to develop kinase inhibitors that are potent as drugs in the treatment of these diseases. Accordingly, kinase assays have played a key role in screening for suitable drug candidates that are kinase inhibitors.
• kinase assays relate to the determination/detection of kinase activity, possibly in order to screen for kinase inhibitor activity of a compound.
• heterogeneous in the context of the above-mentioned assay protocols means that a) product P during the assay is partitioned to a solid phase in an amount that is correlated with the amount of analyte in an original sample, and b) the solid phase containing the immobilized product P, and the liquid phase are separated from each other, i.e. either the liquid phase or the solid phase is removed from the reaction mixture in which immobilization of product P is taking place.
• An analogue of an affinity reactant is capable of competing with and/or inhibiting affinity binding between the affinity reactant concerned and an affinity counterpart to this reactant, e.g. an An-analogue competes with and/or inhibits affinity binding of the analyte to the reactant Re.
• dissolved soluble or “solubilized” are used interchangeable and means that the reactants concerned and product P are true solutes or are in suspended form, for instance firmly attached to suspended particles.
• the liquids discussed herein are typically aqueous, preferably with water as one of the main components of a liquid used (e.g. > 30 % w/w).
• fluid communication between two parts of a microchannel structure means that liquid is intended to be transported between the parts.
• the "ideal" kinase assay should meet the following criteria: a) non-radioactive, b) compatible with both peptide and protein substrates, c) possibility to handle substances that give background fluorescence, d) no negative impact from the reagents used for substrate conversion on the measurement (e.g. luciferase used for measurement may be inhibited by reactants that are present during phosphate transfer), e) possibility to work at high ATP concentrations, f) non-antibody based.
• luciferase used for measurement may be inhibited by reactants that are present during phosphate transfer
• e possibility to work at high ATP concentrations
• f) non-antibody based There is thus a general desire in the field to set up protein kinase assays in which two, three, four, or five of these desires can be met.
• Inefficiency of an assay protocol often depends on poor and/or undesired interactions between dissolved reactants and immobilized affinity reactants, such as immobilized analyte analogues and immobilized components of catalytic systems, or simply between reactants and inner walls of a microchannel structure.
• the latter kind of undesired interactions typically becomes particularly significant and more prominent in nl- volume based microfluidic assays ( ⁇ 1OxIO 3 nl, such as ⁇ 5xlO 3 nl or ⁇ IxIO 3 nl or ⁇ 0.5 ⁇ l ⁇ 3 nl) than in systems utilizing larger volumes ( ⁇ 1 ⁇ l, such as ⁇ 10 ⁇ l or ⁇ 20 ⁇ l).
• nl- volumes primarily contemplate volumes that contain the immobilized reactant or a soluble reactant such as the analyte or an analytically detectable reactant.
• the primary object of the invention is to present solutions that give one or more of the above-mentioned advantages and/or to fully or partly overcome the problems discussed above.
• the present inventors have realized that in order to comply with the primary object of the invention it is appropriate to physically separate the location where the complex (product P) to be measured is formed from the location where the immobilization of product P takes place. By doing so it has been possible to achieve further improvements by selecting immobilization reactions that are fast and/or have equilibriums that suggest non- reversibility under the conditions used.
• the first aspect of the invention is a method for determining the amount of an analyte in a sample by utilizing a microfluidic device of the type generally outlined in the introductory part with the proviso that the formation (step (i)) and measurement (step ii) of product P are physically separated from each other, for instance in RZ (102) and MZ (103), respectively.
• step (i) in some variants means that product P in dissolved form is obtained outside the device.
• the inventive method typically also contains a calculating step (step (Ui)) after the measuring step.
• step (ii) comprises the substeps of: a) immobilizing product P via the immobilizing tag to the solid phase, and b) measuring the amount of product P immobilized to the solid phase.
• the product P formation step includes also introduction of the immobilizing tag on the taggable group.
• the An-analogue comprises a) a first moiety that is related to the analyte and b) a second moiety that is an immobilizing tag or an analytically detectable group, such as a label.
• Reactant Re accordingly contains an analytically detectable group if the An-analogue contains an immobilizing tag, and an immobilizing tag if the An-analogue contains an analytically detectable group.
• the analytically detectable group may in both variants be a label.
• the analyte is typically a low molecular weight compound, for instance with a molecular weight ⁇ 25,000 dalton such as ⁇ 15,000 dalton or ⁇ 10,000 dalton or ⁇
• a lower limit is typically 100 dalton.
• the amount of complex Re — An-analogue formed will correlate with the amount of analyte added and also with the unknown amount of analyte in the original sample. This single step will thus be part of the product P formation step and take place in RZ (102), or outside the device.
• RM reaction microcavity
• the amount of complex Re — An-analogue is in the second step formed in an amount that will correlate with the amount of added analyte and with the unknown amount of analyte in the original sample.
• the second step will be part of the product P formation step and take place in RZ (102), or outside the device.
• two defined liquid aliquots containing of Re (anti-An) and the analyte, respectively, are mixed and incubated with each other in ISA (101) or outside the device (1 st step).
• a defined volume of this mixture is after incubation introduced into RZ (102) where it is mixed and incubated with a defined liquid aliquot containing the non-limiting amount of An-analogue (2 nd step).
• the analyte and An-analogue is allowed to compete with each other for binding to a non-limiting of Re (anti-An) in one single step.
• the preceding step(s) may take place either in ISA (101) of the device or outside device.
• An-analogue or Re comprises a taggable group instead of the immobilizing tag as discussed above, transformation of the taggable group to an immobilizing group is part of the product formation step and takes place either outside the device or within RZ (102), for instance simultaneously with or subsequently to the formation of An-analogue — Re in RM (105) or in a separate reaction microcavity (not shown) that is downstream of RM (105).
• Catalytic assays Catalytic systems primarily contemplate biocatalytic systems, for instance enzymatic systems that are based on enzymatically active proteins or synthetic variants thereof. Components of a catalytic system can be illustrated with catalysts, substrates, cosubstrates, cofactors, cocatalysts, inhibitors, promoters, activators etc including also other effector molecules that are capable of affecting substrate conversion. For enzymatic systems this corresponds to enzymes, substrate, cosubstrates, coenzymes, cofactors, inhibitors, promotors etc.
• the term catalytic system also contemplates coupled systems comprising a catalytic substrate conversion, i.e.
• the product of one system is a component/reactant of another system, e.g. the product or the substrate of an initial catalytic substrate conversion may be a component/affinity reactant of a ligand- receptor affinity reaction system or a reactant in a pure organic/inorganic reaction system.
• the analyte is in this variant of the invention one of the components of the catalytic system at issue (except for not being the product formed by the system).
• the catalytic system should be capable of transforming substrate S to a product P that comprises both the immobilizing tag and an analytically detectable group.
• the detectable group is selected such that it makes it possible to discriminate product P from other entities having tags with the same immobilizing characteristics as product P.
• the catalytic system should be able to produce product P from a substrate S that a) contains the analytically detectable- group but not the immobilizing tag by introducing the immobilizing tag, or b) contains the immobilizing tag but not the analytically detectable group by introducing the analytically detectable group, or c) neither contains the analytically detectable group nor the immobilizing tag by introducing both the tag and the group.
• Alternatives a) and b) typically require single catalytic system while alternative c) typically requires coupled systems (at least one system for the label and at least one for the tag).
• Catalytic systems in the form of enzymatic systems maybe selected amongst: 1) Oxidoreductases (dehydrogenases, oxidases etc), 2) Transferases, 3) Hydrolases (esterases, carbohydrases, proteases etc), 4) Lyases, 5) Isomerases, and 6) Ligases.
• hydrolases for which a number of substrates are known that enzymatically can be transformed to products that have fluorescence or luminescence properties that the corresponding substrates do not have. It would be relatively simple to design this kind of substrates with an immobilizing tag that is retained in the product, for instance by biotinylation or haptenylation.
• a protein kinase is capable of introducing phospho groups on serine and/or threonine and/or tyrosine in protein and polypeptide substrates containing anyone of these amino acid residues.
• the phospho group can be used as a detectable group and measured in step (ii.b) by the use of the appropriate anti-phospho antibody after immobilization via the immobilizing tag in step (ii.a).
• Other transferases are likely to be useful in the analogous manner by the use of the specific groups introduced.
• Ligases may be used in combination with two different substrate molecules that are capable of being ligated by a ligase provided that one of the substrate molecules contains a detectable group while the other one contains an immobilizing tag.
• This mixing typically means that two or more liquid aliquots, each of which contains a single reactant or a combination of reactants, are mixed in the mixing unit.
• One of the aliquots typically comprises at least the analyte.
• components of the catalytic system used are premixed and possibly allowed to bind to each other in mixing units and reaction microcavities upstream RZ (102), i.e. in an RTU (114) of ISA (101), with the proviso that mixtures containing combinations that by themselves leads to formation of product P are only prepared in RZ (102).
• Premixing and/or preincubating in this context contemplate that one or more liquid aliquots containing the remaining components for an active substrate conversion is mixed with the premixture in RZ (102). Suitable remaining components are for instance substrate S, one or more imperative effectors, the catalyst as such etc.
• Premixing and/or preincubation steps, possibly in combination with the actual product P formation step may take place outside the microfluidic device.
• the complete system can be applied in the reaction microcavity RM (105) of RZ (102) as illustrated in the outline of kinase assays in experiment 3.
• individual parts of coupled catalytic system may be applied consecutively with the first catalytic substrate conversion step and subsequent parts of the product P formation step taking place within RZ (102), typically with said substrate conversion taking place within RM (105) and subsequent part steps in one or more reaction microcavities (not shown) that are downstream of RM (105) but within RZ (102).
• Product P is after its formation transported into in the measuring zone (MZ) (103) irrespective of being formed within or outside the device. This applies to both competitive and catalytic assay protocols
• This step comprises two substeps: a) immobilization of the product in the capture microcavity (CM) (106), and b) measurement of the amount of product P that becomes immobilized in CM (106).
• Step (ii.a) and immobilizing tag and a reactive counterpart (anti-tag group)) Immobilization may take place under static conditions or more preferably under flow conditions.
• static conditions in this context contemplates that the flow through CM (106) is halted during immobilization, typically during more than 90 % of the period of time used for contact between product P and the solid phase in CM (106).
• flow conditions contemplates that the reaction mixture containing product P is flowing continuously through CM (106) during immobilization, typically for more than 90 % of the period of time used for contact between product P and the solid phase in CM (106). Flow conditions typically leads to a better concentrating of the immobilized product to the inlet part of the solid phase.
• the flow rate used should give a residence time for the reaction mixture of > 0.010 seconds such as > 0.050 sec or > 0.1 sec with an upper limit that typically is ⁇ 2 hours such as ⁇ 1 hour.
• Illustrative flow rates are within 0.001-10,000 nl/sec, such as 0.01-1000 nl/sec or 0.01-100 nl/sec or 0.1 - 10 nl/sec. These intervals may primarily be useful for solid phase volumes in the range of 1 - 1 ,000 nl, such as 1 -200 nl or 1-50 nl or 1-25 nl.
• Residence time is the time it takes for a liquid aliquot to pass the solid phase. Optimization typically will require experimental testing.
• the liquid flow through the solid phase can be driven by in principle any kind of forces, for instance electrokinetically or non-electrokinetically forces as described elsewhere in this specification. Centrifugal force created by spinning the microfluidic device, possibly combined with capillary force are preferred.
• Immobilization of product P takes place via the immobilizing tag that typically should give selectivity in the immobilization. Constituents (in the liquid in which product P has been formed), which would disturb in subsequent steps, for instance by containing a group that is detectable in the same manner as the analytically detectable group in product P (see below), shouls thus become immobilized to a much lesser degree than product P.
• the solid phase typically contains a firmly attached reactive group that is counterpart (anti-tag) to the immobilizing tag on product P.
• product P will thus become firmly attached to the solid phase via bonds created between the immobilizing tag and the anti-tag group.
• the anti-tag group are in many variants in molar excess in CM (106) compared to the immobilizing tag that is present in the reaction mixture containing product P (e.g. coming from RM (105)).
• the excess may be > 2-fold, such as > 5-fold or > 25-fold or > 50 fold or even more, such as 500-fold or 5000-fold.
• the anti-tag group on the solid phase may be in a deficient amount compared to the immobilizing tag, for instance in the case one would like to reduce the signal from the analytically detectable group, such as the label.
• deficient amount may be ⁇ 0.5-fold, such as ⁇ 0.2-fold or ⁇ 0.04-fold.
• An immobilizing tag and its reactive counterpart (anti-tag) are called an immobilizing binding pair. There are two main kinds of such pairs: a) covalently immobilizing pairs, and b) affinity immobilizing pairs.
• covalently immobilizing pair typically means that the anti-tag group is a chemically reactive group that is capable of forming a covalent bond with the immobilizing tag.
• Typical pairs includes among others so called soft electrophilic groups versus the corresponding soft nucleophilic groups.
• Soft electrophilic groups are ⁇ -halo carbonyl (in particular ⁇ -iodo carbonyl), ⁇ , ⁇ -alkene carbonyls (such as in N-substituted maleimide structures), disulfides (-S-S-) (in particular so called reactive disulfides), and asymmetrically oxidized disulfides (such as -S-SO n - (where n is 1 or 2)), etc.
• the corresponding soft nucleophilic group is primarily the thiol group (-SH). So called hard nucleophilic groups and the corresponding hard electrophilic groups may also be used. Hard electrophilic groups are imido carbonate, oxirane, carbonate etc. Hard nucleophilic groups are hydroxy, amino etc. The electrophilic group is attached to the solid phase in the most typical immobilizing pairs of this kind.
• affinity immobilizing pair means that the anti-tag group is an affinity counterpart to the tag.
• the immobilizing tag is typically called affinity binder or simply binder (B) and its counterpart on the solid phase is called ligand (L).
• affinity binder or simply binder (B)
• ligand L
• This kind of pair should be selected such that, except for the desired affinity binding, the members of the pair should be essentially devoid of other binding abilities during the conditions used.
• the ligand L has two or more binding sites for the binder B 5 and/or binder B has one, two or more binding sites for the ligand L (or vice versa).
• affinity immobilizing 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 IMAC-binding motif (i.e. an oligopeptide containing single or a sequence of histidyl, cysteinyl, phosphorylated aminoacyl etc residues), anti- species specific or anti-class specific antibodies and Ig species specific and Ig class specific determinants etc. Sequence in this context comprises two, three, four, five, six or more residues. Typical aminoacyls that may be phosphorylated are threonine, tyrosine and serine.
• the affinity ligand L should be attached more firmly to the solid phase than the binder B is affinity bound to the affinity ligand L in step (ii.a). This typically means covalent attachment to the solid phase of the affinity ligand L although also adsorptive bonds involving electrostatic attraction, van-der Waals bonding etc may be used.
• Step (ii.a) typically also comprises a separating step in which the solid phase with its immobilized product P is physically separated from excess reactants. Separation is typically accomplished by transporting the liquid phase with dissolved components through the solid phase while immobilizing product P. Components that comprises the tag will be retained by the solid phase while other dissolved components will pass through.
• This separation step may comprise one or more washing steps in which one or more aliquots of washing liquid, are passed through the solid phase to make the removal/separation more efficient.
• Step (ii.b) Measuring the amount of product P in the measuring zone MZ - detectable groups.
• This substep means that the amount of the analytically detectable group that is present in the immobilized product P is measured.
• the reactants and the immobilizing tag have been selected such that the solid phase will contain negligible amounts, if any, of the detectable group that has been immobilized via other routes than via product P.
• the detectable group is typically an affinity group, a signal-generating group or the like.
• the group may be synthetically introduced on a reactant and is then called a label and the labeled reactant is a labeled conjugate.
• the detectable group is natively present in the reactant.
• the analytically detectable group may also be created during the formation of product P .
• detectable affinity groups are biotin, haptens, class-, subclass- or species- specific determinants on antibodies etc.
• Biotin and hapten are examples of compounds that typical are used as labels in the invention, i.e. in conjugates.
• Detectable affinity groups that are present in a product P require as a rule a secondary detectable reactant that has affinity for the detectable group on product P. This secondary reactant comprises a detectable group that should be distinguishable from the detectable group in product P but otherwise is selected amongst the same candidates as the detectable group in product P.
• the secondary reactant preferably is a conjugate and comprises a label that is catalytic, such as a component of a catalytic system, or otherwise is capable of generating a measurable signal (chromophor, fluorophor, luminophor, radioactive etc).
• the detectable group in the secondary reactant may also be a detectable affinity group in which case there is required also a tertiary reactant comprising a detectable group and a moiety that is affinity counterpart to the detectable group of the secondary reactant.
• Secondary, tertiary etc reactants of this kind are preferably introduced via one or more inlet units (107,lll;210) that are in downstream fluid communication with CM (106,206) without passing RM (105,205).
• the introduction may be via other inlet units, e.g. the introduced liquid passing through RM (105,205) in a similar manner as for inlet units (108- 110;207-209).
• the most important signal-generating analytically detectable groups are synthetically introduced into a reactant.
• the generated signal is typically some kind of radiation, such as visible light at a certain wavelength, fluorescence, luminescence, radioactivity etc.
• a signal-generating detectable group may be catalytically active and the detectable group then is a component of catalytic systems such as an enzymatic system.
• the label is typically a catalyst, a co-catalyst, substrate, inhibitor, activator or the like which for enzymatic system will be enzyme, co-enzyme, substrate, co-substrate, cofactor, inhibitor, activator or the like as described for catalytic assays above.
• Catalytically active label typically convert a substrate to a product that may be in dissolved or insoluble form. The substrate differs from the product with respect to one or more detectable properties that are used when measuring the amount of product P.
• a signal-generating group may alternatively be non-catalytic and then typically is a group that is capable of emitting radiation and/or interacting with incoming radiation.
• These labels are typically selected amongst chromophors, fluorophors, luminophors, radioactive groups etc.
• luminophors includes chemiluminophors, bioluminophors and the like.
• the analytically detectable group maybe releasable from the immobilized product P.
• a released label that is soluble (in dissolved form) maybe transported downstream and measured in a separate detection microcavity that is part of the measuring zone MZ (103). See for instance WO 02075312 (Gyros AB) for microfiuidic devices and US 4,231,999 (Carlsson et al) for larger static systems.
• catalytically active labels that result in soluble products, m the case of insoluble products that are analytically detectable, measurement many times can take place directly on the solid phase in CM (106).
• Step (iii). Calculation step.
• This step is performed according to established principles as outlined elsewhere in this specification.
• the main objective is to receive a measure that enables comparison of analyte occurrence and activity between different samples or with standards.
• Amounts in this context contemplate concentrations, such as in absolute and/or relative figures (for instance relative to a standard or to a non-analyte component(s) of the sample etc), and includes binding activity, enzymatic activity etc.
• conjugates primarily refers to man-made/man-designed covalent conjugates between two different affinity binders or between one affinity binder and a label. This kind of conjugates may be obtained by chemically or recombinantly linking the moieties of the conjugates together.
• an-analogue and/or Re are in the form of conjugates.
• conjugates comprise
• A) a first moiety that relative to the analyte in the competitive receptor ligand assays can act as a) an An-analogue, or b) an affinity counterpart (anti-An) to the analyte, or in the catalytic assays can act as substrate S, and
• the two moieties of the conjugates are covalently held together by a bridge that typically is hydrophilic or amphiphilic.
• Typical bridges provide a distance of at least 5 atoms between the moieties. That the bridge is hydrophilic or amphiphilic contemplates that the ratio between the number of heteroatoms (nitrogen and oxygen) and the number of carbon atoms in the bridge is > 0.1, such as > 0.2, and typically is ⁇ 1.
• Instable structures, such as peroxy groups and groups containing a carbon directly binding a hydroxyl or an amino group and an additional oxygen and nitrogen, etc are in most cases not present.
• the second moiety or the bridge is preferably a polymeric carrier for the first moiety or for both in the case the carrier is a bridge.
• Serum albumin and other water-soluble/hydrophilic polymers that do not participate in intended reactions of an assay are a typical polymeric carriers that can be used as a bridging molecule between the first and second moieties. It seems beneficial for An-analogue conjugates to contain a carrier structure as discussed herein for carrying a plurality of An-moiteies, in particular if the other moiety is a label. This kind of conjugates is preferably combined with low-molecular analytes as discussed elsewhere in this specification.
• a microfluidic device is defined as a device in which one or more liquid aliquots that contain reactants and have volumes in the ⁇ l-range are transported and processed in microchannel structures that have a depth and/or width that are/is in the ⁇ m-range.
• the ⁇ l- range is ⁇ 1000 ⁇ l, such as ⁇ 25 ⁇ l, and includes the nl-range that in turn includes the pl- range.
• the nl-range is ⁇ 5000 nl, such as ⁇ 1000 nl.
• the pl-range is ⁇ 5000 pi, such as ⁇ 1000 pi.
• the ⁇ m-range is ⁇ 1000 ⁇ m 5 such as ⁇ 500 ⁇ m.
• a micro fludic device typically contains a plurality of the microchannel structures described above, i.e. has two or more microchannel structures, such as > 10, e.g. > 25 or > 90.
• the upper limit is typically ⁇ 2000 structures.
• Inertia force may be used, for instance by spinning the disc as discussed in the subsequent paragraph.
• Other useful forces are electrokinetic forces and non-electrokinetic forces other than centrifugal force, such as capillary forces, hydrostatic pressure, pressure created by one or more pumps 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 00 ).
• the disc thus may have various polygonal forms such as rectangular.
• the preferred sizes and/or forms are similar to the conventional CD-format, e.g. sizes in the interval from 10% up to 300 % of a circular disc with the conventional CD-radii (12 cm). If the microchannel structures are properly designed and oriented, spinning of the device about a spin axis that typically is perpendicular or parallel to the disc plane may create the necessary centrifugal force for causing parallel liquid transport within the structures. 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 capture microcavity (CM) (106,206) is then typically at a radial position intermediary to two such sections.
• capillary force is used for introducing liquid through an inlet port up to a first capillary valve whereafter centrifugal force or some other non- passive driving means is applied for overcoming the resistance for liquid flow at the valve position.
• centrifugal force or some other non- passive driving means is applied for overcoming the resistance for liquid flow at the valve position.
• the same kind of forces/driving means is also used for overcoming capillary valves at other positions.
• inner surfaces of the parts should be wettable (hydrophilic), i.e. have a water contact angle ⁇ 90°, preferably ⁇ 60° such as ⁇ 50° or ⁇ 40° or ⁇ 30° or ⁇ 20°.
• ⁇ 90° preferably ⁇ 60° such as ⁇ 50° or ⁇ 40° or ⁇ 30° or ⁇ 20°.
• ⁇ 60° such as ⁇ 50° or ⁇ 40° or ⁇ 30° or ⁇ 20°.
• wettability values apply for at least one, two, three or four of the inner walls of a microconduit.
• the wettability or hydrophilicity, in particular inlet arrangements, should be adapted such that an aqueous liquid will be able to fill up an intended microcavity/microconduit by capillarity (self suction) once the liquid has started to enter the cavity/microconduit.
• a hydrophilic inner surface in a microchannel structure may comprise one or more local hydrophobic surface breaks (water contact angle > 90°). Such a break may wholly or partly define a passive/capillary valve, an anti-wicking means, a vent to ambient atmosphere etc. Contact angles refer to values at the temperature of use, typically +25°C, and are static. See WO 00056808, WO 01047637 and WO 02074438 (all Gyros AB).
• Suitable microchan nel structures will be described based on the preferred structure illustrated in figures 2a and b and follow the general outline given in the introductory part.
• the inlet arrangement ISA (101) has one, two or more inlet units (IU) (107-111,207-210), and possible also a reactant or sample transformation unit (RTU) (112-116). Two or more of the inlet units (107-111,207-210) may be in downstream communication with the same part or with different parts of a microchannel structure, for instance with the same microcavity or with different microcavities. Compare IU (210) with IUs (207-209).
• An inlet unit (107-111,207-210) is in the upstream direction typically in fluid communication with ambient atmosphere and therefore comprises an inlet port/opening (217-220) for liquid.
• the inlet unit may also have a volume-defining unit (221-224) in which a liquid aliquot to be transported downstream is metered.
• an inlet unit IU (107-111,207-210) is in fluid communication with the reaction microcavity RM (105,205), the capturing microcavity CM (106,206) and/or some other microcavity in MZ (103), and/or a microcavity in an RTU (112-116).
• volume-defining units (221-224) Metering of liquid volumes in volume-defining units (221-224) is preferably based on the over-flow principle.
• a detailed description of the preferred volume-defining unit given in figure 2 is given in WO 02074438 (Gyros AB).
• the corresponding inlet unit in several microchannel structures (subgroup) of a device may be linked together in a liquid distribution manifold that is common for the microchannel structures. See WO 02074438 (Gyros AB).
• a reactant and sample transformation unit (RTU) (112-116) comprises functional units that are necessary for transforming samples and reactants that are introduced into the structure to forms that are required by the product P formation step in RM (105,205) or other process step as described in this specification. Typical such functional units are separation units for removing undesired particles, mixing units, volume-defining units, reaction microcavities etc. Different RTUs are connected to the same or to different parts of a microchannel structure, for instance one RTU may be connected to RM (105,205) and another one to CM (106,206), respectively.
• An RTU (112-116) is typically connected to ambient atmosphere via an inlet unit (107-111) for liquid.
• Suitable inlet units including liquid distribution manifolds are described in WO 04058406 (Tecan), WO 9853311 (Gamera Biosciences), WOOl 87486 (Tecan Trading), WO 02074438 (Gyros AB), WO 03018198 (Gyros AB), WO 9958245 (Amersham Pharmacia
• a reaction zone RZ (102) comprises one or more reaction microcavities used for carrying out the product P formation step.
• the most upstream of them RM (105,205) is shown in figure 2 and is used for the formation of the complex An-analogue — Re in an amount that is related to the analyte (protocol (a)) or for the substrate S conversion step (protocol (b)).
• Other reaction microcavities in RZ (102), if present, are located downstream of RM (105,205) and are typically used for further processing the resulting reaction mixture obtained in RM (105,205), for instance removal of entities that otherwise may have a negative impact on the measurement or immobilization in MZ (103) and derealization of the product formed in RM (105,205).
• Derivatization in this context may comprise that a product comprising an analytically detectable affinity group is reacted with a labeled conjugate that comprises an affinity counterpart to this particular detectable group, or that a reactant comprising an immobilizing tag is reacted with the product obtained in RM to introduce the tag on the product.
• a reaction microcavity RM (105,205) in RZ (102) comprises one, two or more inlet openings (225-226) and at least one outlet opening (227). Each inlet opening (225-226) is connected to an inlet microconduit (228-229) and the outlet opening (227) to an outlet microconduit (230).
• the inlet microconduit (228-229) is in the upstream direction in fluid communication with a vent function and/or one or more inlet units (108-110,207-209) for liquid as discussed above possibly via various parts of ISA (101) or via a mixing function (231) used for mixing the liquid aliqu ⁇ ts used in the product P formation step in RM (205).
• the outlet microconduit (230) is in the downstream direction in communication with CM (206) of MZ (103).
• RM (205) is typically associated with a mixing unit (231), which for instance comprises a mechanical mixer or is based on mixing during liquid transport in a mixing microconduit that ends in a collection microcavity (that typically also is used as a reaction microcavity) as recently suggested for centrifugal based microfluidic devices) (US 6,582,663, WO 00079285, US 6,527,432, US 20020097632; US 20030152491, WO 01087487 (all Tecan Trading); WO 02074438, WO 03024598, WO 05094976 (all Gyros AB)) and for non- centrifugal based microfluidic devices (US 6,379,929 (Univ. Mich.)).
• a mixing unit (231) for instance comprises a mechanical mixer or is based on mixing during liquid transport in a mixing microconduit that ends in a collection microcavity (that typically also is used as a reaction microcavity) as recently suggested for centrifugal based
• This mixing unit may fully or partly coincide with RM (205) and/or with one or more of the inlet microconduits (228-229) of RM (205). See figure 2. Compare also the mixing function in WO 02074438 (unit 2) (Gyros AB) and WO 03018198 (units A and B (Gyros AB). The variant illustrated in figure 2 utilizes back-and-forth transport in the mixing microconduit of the aliquots to be mixed. See WO 05094976 (Gyros AB).
• One or more of the additional reaction microcavities in RZ may or may not be associated with a mixing function as discussed for the mixing function (231) associated with RM (205).
• the measuring zone MZ The measuring zone MZ
• the MZ (103) comprises at least a capture microcavity CM (106,206) that contains a solid phase to which product P is to be immobilized.
• the measuring zone MZ (103) also comprises a detection microcavity DM that may coincide with CM (206) (as in figure 2) or is placed downstream CM (not shown).
• a detection microcavity DM that coincides with CM (206) is used when the analytically detectable group that generates the signal to be measured is retained on the solid phase.
• a detection microcavity DM that is separate from CM (106,206) is primarily used when detecting/measuring the immobilized product P utilizes a soluble signal generating substance that is formed in, released from, or passed through and partly consumed in CM (206).
• This kind of detection microcavity DM may comprise a solid phase for capture of the signal generating substance and detection/measurement on the solid phase, or is devoid of a solid phase meaning that detecting/measuring of the signal-generating substance is taking place in solution.
• CM and DM there is preferably a liquid split (router, branching) permitting waste liquids to pass to a waste function without passing through DM.
• WO 0274438 Gyros AB
• WO 05032999 Gyros AB
• US 2005032999 Gyros AB
• CM (106,206) typically comprises an anti-tag group that is used for the immobilization of product P.
• CM (106,206) is typically a straight microconduit that may or may not be widening and/or narrowing in the flow direction.
• the capture microcavity CM (106,206) typically has at least two upwardly directed microconduits (232-233) and one or more microconduits (234) that at least initially are directed downwards.
• One of the upwardly directed microconduits is a liquid inlet microcoduit (232) through which product P is introduced into CM (106,206) and thus is in upstream fluid communication with the reaction microcavity RM/reaction zone RZ (105,205/102), if present.
• the remaining upwardly directed microconduits (233) are typically used for venting purposes and/or for separate introduction of liquid aliquots that are not needed in the product P formation process. If an upwardly directed microconduit of the latter kind is used for inlet of liquid into CM (106,206) it is typically in upstream fluid communication with an inlet unit IU and/or reactant and sample transformation unit RTU, such as inlet ports and volume-defining units, respectively, that is separate from the corresponding units for liquid aliquots containing reactants used in the formation of product P.
• an inlet unit IU and/or reactant and sample transformation unit RTU such as inlet ports and volume-defining units, respectively
• CM is part of an Y-shaped structure as illustrated in figure 2 with two upwardly directed liquid inlet microconduits (232-233) and one downwardly directed outlet microconduit (234) and with CM (106,206) located downstream of the merging of the two inlet microconduits.
• the CM typically has at least one cross-sectional dimension in the ⁇ m-range.
• the volume is typically in the nl-range.
• the outlet microconduit (234) from CM (206) is typically designed as a restriction microconduit that is capable of creating a pressure drop that is larger than the total inter- channel variation in flow resistance emanating from positions upstream and downstream the microconduit. See further WO 03024598 (Gyros AB).
• the solid phase may be a porous bed, i.e. a porous monolithic bed or a bed of packed particles that may be porous or non-porous.
• the solid phase may be an inner wall of CM (206).
• CM CM
• the term "porous particles” is given in WO 02075312 (Gyros AB).
• Suitable particles to be used in a porous bed are spherical or spheroidal (beaded), or non- spherical.
• Appropriate mean diameters for particles 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. Diameters refer to the ' "hydrodynamic" diameters.
• Particles may be monodisperse (monosized) or polydisperse (polysized) in the same meaning as in WO 02075312 (Gyros AB).
• 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).
• 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 an aqueous liquid medium during the immobilization 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, 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 pH 2-12.
• 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 to contain polar functional groups of the same type as discussed above.
• the solid phase is typically predisposed to CM (106,206) by which is meant that the solid phase is introduced into the microchannel structure before the actual assay protocol is performed. Predisposing may thus take place before the device is offered for sale, for instance during the manufacture of the device. See for instance WO 04083108 (Gyros AB) that among others describe predisposed solid phases exposing a firmly attached affinity counterpart to an immobilizing tag.
• CM (106,206) may be connected to one or more separate or joined inlet units (207,210 and
• inlet units are in the downstream direction uniquely associated with CM (106,206) or with positions in the measuring zone that are upstream of CM. Their connections to the measuring zone (103) thus do not pass through RM (105) or RZ (102). Between this kind of inlet units and the measuring zone there may be a RTU (not shown). This RTU may contain one or more units selected from separation units, volume-defining units, reaction microcavities, mixing units etc as generally discussed above for ISA (101) and RTU (116). This kind of inlet units is preferably used for the introduction of reagents and washing liquids used in the measuring step. See above.
• the microchannel structure typically contains a number of valve functions and other functions controlling the liquid transport.
• a valve 235-238,239,240-243 at the outlet opening of each volume-metering microcavity (244-247), at the outlet opening (227) of each reaction microcavity (205) and in the downstream part of each overflow microconduit (248-251).
• These valves may be mechanical or non-mechanical including non-closing valves such as passive valves or capillary valves.
• Preferred capillary valves are based on abrupt changes in at least one cross-sectional dimension and/or an abrupt non- wettable break (hydrophobic break) in the wettability of an otherwise wettable microconduit.
• the microchannel structure typically also contains a number of vents (252-258) for inlet and/or outlet of ambient atmosphere in- order to promote smooth liquid transport without creation of local overpressures and gas bubbles.
• wicking At the appropriate positions within a microchannel structure there may also be so called anti-wicking functions to prevent undesired transport in the edges by wicking, for instance between different functionalities and/or in inlet units to render losses by evaporation difficult.
• Capillary valves (235-238,239,240-243) based on local non-wettable breaks in wettable surfaces may often work as anti-wicking functions Details of vents, inlet ports, anti-wicking functions etc are discussed in WO 9721090
• the micro fluidic device used for examples 1 and 2 is circular and of the same dimension as a conventional CD (compact disc).
• the microstructures of the disc are illustrated in figure 3.
• Each group (359) comprises a distribution manifold/inlet unit (307) that is common for all eight structures of the group plus one inlet unit (308a-h) and one capture microcavity (306a-h) per microchannel structure.
• the common inlet unit (307) comprises a) two common inlet ports (317a-b) that also will function as outlet ports for excess liquid, and b) one volume-metering microcavity (344a-h) for each microchannel structure (300a-h).
• Each of the separate inlet units (308a-h) comprises an inlet port (318a-h) and a volunie- defining unit (321 a-h) in which there is a volume-metering microcavity (345a-h).
• each volume-metering microcavity there is a passive valve function (335a-h,336a-h).
• Each capture microcavity (306a-h) is in the downstream direction directly connected to a narrow outlet microconduit (334) (restriction microconduit).
• this outlet microconduit (334) is designed as an outward bent and is connected to OWA (304).
• capillarity By applying the appropriate volume of aqueous liquid to the inlet port of an inlet unit, capillarity will fill the volume-metering unit(s) connected to the inlet port with liquid.
• liquid By spinning the disc around its center, liquid can be forced to pass the passive valve (335a- h,336a-h) at the outlet of the corresponding volume-metering microcavities for transport through the capture microcavities/solid phases (306a-h).
• 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.
• 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.
• Figure 4 Quantitative assay of substance P performed according to the invented method in a microfluidic device (CD).
• Substance P standards were mixed with anti- substance P and incubated with substance P standards (0-2000 pg/ml) and Substance P-ALP conjugate.
• Reaction products were captured in affinity columns (anti-rabbit IgG) in the CD.
• the columns were equilibrated with chemiluminescent alkaline phosphatase (ALP) substrate (Lumiphos 530TM; Lumiphos 530TM is a trademark of Lumigen Inc., Soutb ⁇ eld , Mi, USA; cat.# 80-0134 Assay Designs, Inc. MI, USA) and the intensity of light emitted from the columns were recorded with a luminescence detector. Samples were analyzed in triplicates.
• ALP chemiluminescent alkaline phosphatase
• the reaction mixture contained phosphate buffered saline (PBS) pH 7.4 and 0.1 % (w/v) bovine serum albumin.
• PBS phosphate buffered saline
• MTP microtitre plate
• biotinylated NPY/antibody was captured in the streptavidin coated beads packed as columns in the CD microfluidic device in the same manner as in experiment 1.
• the column was then treated with Alexa647-labeled goat anti rabbit IgG antibody solution (1 : 500 dilution) and subsequently washed with PBS buffer containing 0.01 % (v/v) Tween20.
• Captured and concentrated biotinylated NPY/antibody complex was quantified by means of a laser induced fluorescence detector. The result for a series of standards is given in figure 5.
• the CD liquid processing steps and the measurement was performed in the same instrument.
• Figure 5 Quantitative assay of human neuropeptide Y according to experiment 2. Anti- NPY (InM) was mixed and incubated with NPY standards (0-10000 nM) and biotinyl- NPY. Samples were analysed in duplicates.
• FIG. 6 Schematic description of the assay methodology used in experiment 2. First biotinylated analyte (tracer) analyte (sample) and Alexa labelled antibody are mixed and incubated. Subsequently, biotinylated analyte/ Alexa labelled antibody complex is captured on streptavidin coated particles packed in a column. Fluorescence-analysis of the column allows for quantification of the biotinylated analyte/ Alexa labeled antibody complex.
• EXPERIMENT 3 Procedure for kinase assay in a CD: Below follows a schematic description of how kinase assays may be performed in a microfluidic device comprising the microchannel structure given in figure 2 with commercially available reagents. The method is based on the use of a biotinylated substrate that may be a peptide or a protein. The method uses an antibody directed towards a phosphorylated substrate (product P). For tyrosine kinase assays anti-phoshotyrosine specific antibodies are commercially available. They recognize all phosphorylated tyrosines, regardless of the amino acid surrounding them.
• Reagent Biotinylated peptide (or protein) + Alexa labeled anti- phosphotyrosine (or anti-phospho-serine/threonine) + ATP+Mg 2+ .
• reaction mixture is spun down over the down stream column/solid phase placed in the capturing microcavity (206).
• Biotinylated peptide/Alexa labeled antibody complex is captured in the streptavidin-columns.
• the reaction product can be quantified my means of a LIF detector by integrating the fluorescence signal in the column.
• Kinase inhibitor screening in CD The proposed assay strategy for kinases described above may also be applicable for inhibitor screening. Importantly, such assays should be possible to perform by mixing only two solutions.
• Classically the assay principle involves an enzyme-catalysed reaction that is allowed to proceed in the presence of different drug candidates at various concentrations. Each substance is tested at different concentration to determine the inhibition mechanism and inhibition constant. In this type of experiments it is likely that the user has hundreds or thousands of drug candidates (inhibitors) stored as stock solutions in MTP:s.
• "Small molecule” organic substances are usually dissolved in a water mixable organic solvents such as DMSO or acetonitrile.
• the "drug” candidates can be considered to be “expensive" reagents as their synthesis often involves many manual steps. Below follows a general assay description.
• Reagent Biotinylated peptide (or protein) + Alexa labeled anti- phosphotyrosine (or anti-phospho-serine/threonine).

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