WO2013090107A1 - Fluorescent measurement in a disposable microfluidic device, and method thereof - Google Patents
Fluorescent measurement in a disposable microfluidic device, and method thereof Download PDFInfo
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- WO2013090107A1 WO2013090107A1 PCT/US2012/068110 US2012068110W WO2013090107A1 WO 2013090107 A1 WO2013090107 A1 WO 2013090107A1 US 2012068110 W US2012068110 W US 2012068110W WO 2013090107 A1 WO2013090107 A1 WO 2013090107A1
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- microns
- luminal surface
- dye
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- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- 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
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N21/03—Cuvette constructions
- G01N2021/0325—Cells for testing reactions, e.g. containing reagents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N21/03—Cuvette constructions
- G01N21/05—Flow-through cuvettes
Definitions
- the present invention relates to the quantitative optical detection of target biological ana!ytes of the type in a biological specimen, such as a patient body fluid.
- the present invention is more specifically related to a device and method for achieving a true and specific optical signal emitted from fluorescently labeled target anal tes.
- the true and specific optical signal is achieved by attenuating the interfering fluorescence emitted from fluorescent detector molecules that are non-specifically bound to the luminal surface of an assay chamber.
- the optical signal accurately reflects the concentration of the target anaiyte in the biological specimen when assayed in an assay chamber of a microfluidic device according to the invention described herein.
- Fluorescent measurement of a target anaiyte in biomedical assays may be conducted in an assay chamber in which one portion of the chamber has an optically clear surface that is coated with binding partners specific for a target anaiyte of interest in a biological sample,
- cells are grown on the optically clear luminal surface of a ceil assay vessel.
- ceil based assays the vessel must be sufficiently large, i.e., capable of holding sufficient fluid (often greater than 100 microliters), to maintain the cells with appropriate needs such as nutrition, oxygen, and waste removal.
- the optically clear luminal surface of such cell-based assay vessels is specifically treated to allow the cells to attach to its luminal surface,
- Other luminal surfaces of the cell assay vessel are treated with blocking agents to minimize non-specific binding to these other luminal surfaces.
- Cell membrane potential changes for example, may be assayed based on fluorescence changes of membrane potential-sensitive dyes which interact with the cells to emit fluorescent signals. Fluorescence is optically measured by an optical detector through the optically clear luminal surface, typically the bottom surface, of the vessel.
- the biological sample suspected of having the target anaiyte of interest typical ly is mixed in a solution with fluorescent detector molecules having a binding partner that is specific for and binds to the target analyte.
- the biological sample with the target analyte of interest bound to the iluoresceni detector molecule flows as a solution into the lumen of an assay chamber having a portion thai is optically clear.
- the luminal surface of the optically clear portion is coated, with binding partners of the target analyte.
- the target analyte in the biological sample binds to the binding partner on the optically clear surface bringing with it the iiuorescent detector molecule.
- the biological specimen is introduced (with or without first mixing with an appropriate assay reagent) into the assay chamber such that any analyies will, be specifically bound to the binding partners on the optically clear luminal surface.
- the chamber is washed to remove unbound, analyte and specimen components and refilled with fluorescent detector molecules.
- the chamber is again washed to remove unbound fluorescent detector molecules.
- a typical problem encountered in biomedical assays of the above types is non-specific binding of iluoresceni detecior molecules to luminal surfaces of the chamber.
- Such non-specific surface binding may occur directly or indirectly by iiuorescent detector molecules complexing with a biological moiety found in the sample, for example, a protein.
- the complex binds to luminal surfaces of the assay chamber other than to the binding partner-coated luminal surface of the optically clear surface of the assay chamber.
- non-specific binding and background fluorescence adulterates the actual fluorescent signal emitted from the target analyte obscuring the optical, signal that would otherwise accurately reflect the quantity of target analyte in a biological sample, such as a patient body fluid.
- the present invention is directed to automated, cost-effective, high throughput solutions that minimize background fluorescence of detector molecules bound non-speeifically to luminal surfaces of an assay chamber, while avoiding the problems and cost associated with blocking non-functionalized chamber luminal surfaces, in particular, background fluorescence arising from the luminal surface opposite an actively treated optically clear surface Is substantial ly reduced, without attenuating the optical signal originating from the target analyte bound to the optically clear activated surface.
- the assay for measuring a specific target anaiyie as defined by the invention is conducted in a micro fluidic device which permits extremely rapid test results while simultaneously improving assay sensitivity, and accuracy and minimizing the expenditure of costly reagents.
- the invention relates to device, kit, or a composition of matter for achieving a true and specific optical signal emitted from fluorescently labeled target biological analytes in an assay chamber.
- the invention includes a niicrofluidic device having an assay chamber for detecting a target analyte.
- the assay chamber includes a first wall with at least a portion of the first wall being optically clear, an opposite wall, and a lumen.
- the entire first wal l is optical ly clear.
- the first wall is coated on the luminal surface with binding partners specific for a target analyte in the biological sample.
- the luminal surface of the opposite wail may be coated or, optionally, uncoated with binding or blocking agents
- the device, kit, or composition of matter includes a fluorescent detector molecule comprising a binding partner for the target analyte, a solution in the assay chamber comprising a dye which is capable of absorbing the light of a wavelength range selected from the group consisting of emission wavelength range, excitation wavelength range, or their combination, of any fluorescent detector molecule that is non-speeiiically bound to the luminal surface of the chamber,
- the dye may be a single standard dye selected from the group amaranth, brilliant green, erioglaueine, for example, or a combination of standard dyes.
- the binding partner that is coated on the luminal surface of the first wall or just the optically clear portion of the luminal surface of the first wall comprises an antibody specific for the target analyte.
- the binding partner of the fluorescent detector molecule comprises another antibody specific for the target analyte.
- the binding partners that are coated on the luminal suriace of the first wall may comprise an intermediate binding partner,
- the distance between the first wall and the opposite wal l is in the range of about 10 microns to 5,0 millimeters, about 75 microns, about 50 microns to 200 microns, or about 70 microns to 100 microns.
- the composition, kit, or device includes fluorescently labeled target analyte molecules.
- fluorescently labeled target analyte molecules may be useful in a competitor binding assay.
- the invention in another aspect, relates to a method for attenuating non-specific fluorescence in a niicrofl idic device used to measure fluorescently labeled target analytes in a biological specimen.
- a sample is introduced into the chamber lumen of the microfluidic device described above.
- a fluorescent detector molecule comprising a binding partner for the target analyte is introduced into the chamber lumen.
- the chamber lumen may be washed.
- the volume of wash solution may be less than, the same as, or greater than the volume of the chamber lumen.
- a solution comprising an attenuating dye for example, amaranth, erloglaucine, brilliant green, or combinations of standard dyes, is introduced into the chamber,
- the dye is capable of absorbing light of a wavelength range selected from the group consisting of emission wavelength range, excitation wavelength. range, or their combination of any fluorescent detector molecule that is non-speeifieally bound to the luminal surface of the chamber.
- An optical measurement Is made and is related to the target analyte concentration in the sample.
- Optically measuring comprises measuring an optical signal arising from the luminal surface of the first wall.
- the method of the invention is a competitive binding assay including the step of introducing iiuorescently labeled target analyte molecules into the chamber lumen.
- the sample and fluorescent detector molecule comprising a binding partner for said target analyte are mixed together before introducing the sample and the fluorescent detector molecule into the chamber.
- the lumen of the chamber is washed after introducing the sample into the chamber lumen and prior to introducing the fluorescent detector molecule into the chamber lumen.
- the lumen of the chamber may be washed with a wash reagent before introducing the dye.
- the lumen of the chamber is washed with a wash reagent containing the attenuating dye
- the volume of the wash reagent is the same as or exceeds the volume of the chamber, in one embodiment, the washing step introduces a wash reagent through an inlet port of the chamber and removes the wash reagent through an outlet port, of the chamber.
- the non-specifically bound fluorescent detector molecule according to the method of the invention is coupled to another molecule, e.g.. a non-target analyte.
- FIG. 1A is a plan view of an exemplary instrument system including a microfluidic device according to one embodiment of the invention.
- FIG. I B illustrates a top cutaway view of an exemplary assay chamber according to one embodiment of the invention.
- FIG. 1C illustrates a bottom cut away view of the exemplary assay chamber illustrated in FIG. IB.
- FIG. I D illustrates a top cut away view of another exemplary cylindrical assay chamber according to one embodiment of the invention.
- FIG. IE illustrates a bottom cut away view of the exemplary cylindrical assay chamber illustrated in FIG, ID.
- FIG, IF illustrates a top view of an exemplary assay chamber and method of making according to one embodiment of the invention.
- FIG . 2 is a diagrammatic cross-sectional view of an assay chamber without attenuating dye.
- FIG. 3 is a diagrammatic cross-sectional view of an exemplary assay chamber including an attenuating dye according to one embodiment of the invention
- FIG. 4 is perspecti ve view of an exemplary assay chamber including an optical signal portion of a wall according to one embodiment of the invention.
- microfluidic device shall mean devices for biological assays that utilize fluid volumes on the order of plcoliters to microliters.
- the devices have channels and/or chambers with dimensions ranging from millimeters to micrometers.
- target biological analyte shall mean an anal vie or a group of analytes of interest in a biological specimen such as but not limited to pathogens, proteins, nucleic acids, lipids, antibodies, an tigens, and enzymes.
- a group of analytes may he a plurality of proteins, .for example, myoglobin, proBNP, and myosin, proteins that are useful in detecting heart failure.
- a fluorescent detector molecule shall mean any molecule, binding partner, or entity tha can complex directly or indirectly with another molecule or substance and can be detected using a suitable fluorescence optic system, wherein the molecule, binding partner or entity is excited by light of an appropriate wavelength and the emitted light (at a different wavelength) is measured.
- the molecule, binding partner or entity may be intrinsically fluorescent or rendered fluorescent by attachment of an appropriate fluorophore,
- an attenuating dye shall mean a dye that absorbs light of a wavelength range including emission wavelength range, excitation wavelength range, or the combination of emission wavelength range and excitation wavelength range of any fluorescent detector molecule.
- a binding partner shall mean a molecule, for example, an antibody which binds specifically to a target biological analyte, or an intermediate in a binding cascade, for example, where strepavidin is coated onto a surface as an intermediate binding partner, and the strepavidin then binds to biotin which has been conjugated to an antibody that is a specific binding partner for a target biological analyte.
- background fluorescence shall mean fluorescence that has not originated from a fluorescent detector molecule bound to a target analyte of interest
- the invention relates to a disposable microfluidic device for optical measurement of a target biological analyte in a biological specimen such as, but not limited to, body tissues, or a patient body fluid, for example, blood, serum, plasma, urine, sputum, cerebrospinal fluid, joint fluid, digestive fluid, tissue aspirates, exudates, and transudates.
- a biological specimen such as, but not limited to, body tissues, or a patient body fluid, for example, blood, serum, plasma, urine, sputum, cerebrospinal fluid, joint fluid, digestive fluid, tissue aspirates, exudates, and transudates.
- Embodiments of the invention relate to an apparatus, kit, composition of matter, or method, for example, an immunoassay method, for detecting target, anaiytes in an assay chamber of a microfluidic device.
- Figures 1 A-F are exemplary embodiments of a disposable microfluidic device and instrument system according to the invention thai has been developed for sensitive, accurate, cost-effective, and automated diagnostic testing of a target analyte of interest and generates rapid test results.
- the instrument system includes a microfluidic device 9 having an assay chamber 10 and fluid conduits 2, a microfluidic device holder 4, microprocessor 6, electronics 8, and an optical system 92 comprising an optical source 90 and an optical detector 100 for measuring optical signals such as optical signals generated by a fluorescent detector molecule bound to a target analyte in an assay chamber.
- the microfluidic device includes a rectangular assay chamber 10 which has 6 walls 12 n , specifically, 12a, 12b, 12c, 12d, 12e, and 12f, surrounding a chamber lumen 16.
- the assay chamber 10 is capable of holding a fluid when any wall could be the wail closest to the source of gravitational pull.
- the chamber 10 is completely enclosed on all sides with the exception of optional ports, for example, inlet or outlet ports, in one embodiment, the chamber 10 may be a channel with optional inlet and/or outlet ports at the channel ends.
- the shape of the chamber 10 of the microfluidic device is not limited by the shapes illustrated in the appended figures.
- Each wall 12a ⁇ 12f of the chamber 10 has a luminal surface 14 adjacent the lumen 16.
- the chamber 10 has an inlet port 20 and an outlet port 22.
- An active, optically clear wall portion is positioned within wall I 2f, or optionally, as illustrated in FIG. IB, the entire wall 12f is optically clear.
- the luminal surface 14f of the wall 12f, or optionally only the optically clear portion of wall 12f is activated by coating the surface with binding partners specific for a target analyte of interest,
- the wails 12d and 12f may be planar or may have one or more radii.
- the chamber wall 12d that is opposite to the optically clear wall 12f is substantially parallel to, 0 to 45 degrees, 0 to 10 degrees, or 10 to 45 degrees, for example, relati ve to the plane of the optically clear wall 12f.
- the luminal surface of chamber wall 12d is substantially parallel to, 0 to 45 degrees. 0 to 10 degrees, or 10 to 45 degrees, for example, relative to the plane of the luminal surface of optically clear wall 12f
- the luminal surface 14 of the chamber walls 12a-12e other than the luminal surface 14f of the optically clear wall 12f are uncoated with binding partners or with blocking agents or any other agents prior to initiation of an assay that would otherwise block non-specific binding to the luminal surfaces of these walls.
- the assay chamber 10 may be made from a polymer, for example, but not. limited to, polystyrene.
- the assay chamber 10 is substantially rectangular with an optically clear wall 12f (or portion thereof) and a wall 12d opposite the optically clear wall 12f.
- the distance 80 between the luminal surface I4f of the optical ly clear wall 12f and the luminal surface 14d of the wall 12d opposite the optically clear wall 12f is in the range of about 10 microns to 5 millimeters, 10 microns to 2 millimeters, 10 microns to 1 millimeter, 50 microns to 200 microns, 50 microns to 125 microns, 70 microns to 100 microns, 75 microns to 150 microns, preferably 50 to 100 microns, more preferably 75 microns.
- the chamber lumen 16 is bounded and enclosed by the walls 12a-12f including the optically clear wall 12f and the wail 12d opposite the optically clear wall of the chamber 10.
- the walls other than the optically clear wall may be made from a light blocking material, for example, a black plastic. Alternatively, the walls may be optically clear.
- assay chamber 1 0 is substantially cylindrical with wall 12f and wall 12d at opposite ends of the cylindrical chamber 10. and wall 12b joining wall 12f and 12d, Wall 12f of the chamber 10 is optical Sy clear or, optionally, a portion of wall 12f is optically clear.
- the chamber wall 12d that is opposite to the optically clear wall 121 " is substantially parallel, 0 to 45°, 0 to 10°, or 10 to 45° relative to the plane of the optically clear wall 12f.
- the luminal surface of chamber wall 12d is substantially parallel, 0 to 45 degrees, 0 to 10 degrees, or 10 to 45 degrees, for example, relative to t he plane of the luminal surface of optically clear wall 12f.
- the luminal surface 14f of the optically clear wail 12f or a portion of the luminal surface wail 12f of the cylindrical chamber 10 is activated by coating the surface with binding partners specific for a target analyte of interest by standard methods known to the skilled artisan.
- the luminal surface of the walls 12b and 12d are uncoated with binding partners or with blocking agents or any other agents prior to initiation of an assay that would otherwise block non-specific binding to the luminal surfaces of these walls.
- the distance 80 between the luminal surface 14f of the optically clear wall 12f and the luminal surface 14d of the wall 12d is in the range of about 10 microns to 5 millimeters, 10 microns to 2 millimeters.
- microns to 1 millimeter 50 microns to 200 microns. 50 microns to 125 microns, 70 microns to 1 0 microns, 75 microns to 150 microns, preferably 50 to 100 microns, more preferably 75 microns.
- the chamber may assume other shapes (e.g. shapes with curved side portions as opposed to orthogonal edges may facilitate optimal fluidic properties when introducing and removing solutions from the chamber), a channel for example, and is not limited to the illustrated rectangular or cylindrical shapes.
- the walls other than the optically clear wall may be made from a light blocking material, for example, a black plastic. Alternatively, the walls may be optically clear.
- a first chamber part is a shallow well 40 made from a polymeric material and having a wall 12d at the bottom of the shallow well 40, an open face 42 at the top of the shallow well, and well side walls 12a, 12b, 12c and 12e.
- the shape of the well 40 is not limited to rectangular but may be oval, circular, or other shapes, for example.
- the depth of the shallow well 40 is in the range of about 10 microns to 5 millimeters, 10 microns to 2 millimeters, 10 microns to 1 millimeter, 50 microns to 200 microns, 50 microns to 125 microns, 70 microns to 100 microns, 75 microns to 150 microns, preferably 50 to 100 microns, more preferably 75 microns.
- the optically clear wall 12f, or optionally, a portion of wall 12f of the assay chamber 10 is activated by coating the surface on one side of the wall with binding partners, defined above, for the target analyte of interest (see, e.g., FIG. 2),
- the binding partner coated on the surface may be, but is not limited to, for example, polyclonal or monoclonal antibodies and fragments thereof specific for a target analyte, other proteins, lectins, antibodies,
- the coated surface of the optically clear wail 12f is placed face down on the open face 42 of the shallow' polymeric wel l 40 such that the coated surface is on the luminal side of the newly formed chamber 10.
- the optically clear wall 12f is affixed to the top of the walls of the shallow polymeric well 40 by adhesives, heat bonding, ultrasonic welding, or other methods of permanent attachment,
- the luminal surfaces 14 of the shallow well portion 40 of the chamber 10, including the luminal surface 14d of the wall 12d at the base of the shallow well 40 are not treated with any agents prior to initiation of an assay, such as blocking agents, for example, but not limited to the blocking agents casein, bovine serum albumin, and newborn calf serum.
- chamber 10 as described above, is readied for an assay.
- Chamber 10 is fi lled with the biological sample suspected of having the target analyte of interest.
- the chamber lumen 16 is washed by introducing a volume of wash solution through the inlet port 20 that exceeds or is equal to the volume of the chamber lumen.
- the wash solution may be removed through outlet port 22,
- the fluorescent detector molecules with binding affinity for the target analytes are added to the chamber lumen and incubated for sufficient time to allow binding to occur.
- the chamber is again washed prior to optical detection to remove unbound fluorescent detector molecules.
- the fluorescent detector molecules with binding affinity for the target of interest may be pre-mixed with sample, The mixture is then introduced into the chamber, followed by washing the chamber lumen, which is followed by optical detection.
- the binding partners of the fluorescent ⁇ detector molecules that are mixed with the biological sample are different than the binding partners for the target anaiyte coated on the luminal surface of the optically clear wail,
- the binding partners integral to the fluorescent detector molecules and the binding partners coated on the luminal surface may be the same, for example, when the target anaiyte is multivalent.
- the binding partners may be purposefully designed to bind to a group of closely related target analytes, for example to detect all members of the distinct, but closely related subtypes of HIV viruses.
- the binding partners may be intermediates in a binding cascade, for example where streptavidin.
- Streptavidin then binds to biotin which has been conjugated to an antibody that is specific for the anaiyte of interest,
- the target anaiyte in the sample binds to the binding partner of the fluorescent detector molecules when the target anaiyte and binding partner are contacted in solution, thereby forming a fluorescently labeled target anaiyte.
- excitation light from an optical source 90 of the instrument system is directed through the optically clear wall 12f or a portion of the optically clear wall 12f of the assay chamber 10 to excite fluorescence 56 of the fluorescent detector molecules 52 bound to the target analytes 55 which in turn are bound to the binding partners 57 on the luminal surface 14f of the optically clear wall 12f.
- Fluorescence 50 detected by an optical detector 100 from fluorescent detector molecules 52 non-specifically bound to the untreated luminal surfaces of portions of the assay chamber, the opposite wall luminal surface 14d in particular, is unwanted background fluorescence.
- the background fluorescence 50 overlaps the fluorescence 56 emitted from the target anaiyte bound 55 to the binding partners 57 on the luminal surface 14f of the optically clear wall 12f of the assay chamber 10.
- the optical signal received by the optical detector includes a backgro und contribution that is not related to the concentration of the target analyte.
- the efficiency of fluorescence excitation and collection from the non- treated luminal surface 14d of the opposite wail 124 of the assay chamber 10 and the activated luminal surface 14f of the optically clear wall 12f is essentially identical given the narrow distance 80 (in one embodiment described above, as little as 10 microns) between the luminal suriace 14f of the optically clear wail 12f and the luminal surface 14d of the wai l 12d opposite to the optically clear wail.
- the active surface is merely about 10 microns to 5 millimeters, 10 microns to 2 millimeters, 10 microns to 1 millimeter, 50 microns to 200 microns, 50 microns to 125 microns, 70 microns to 100 microns, 75 microns to 150 microns, preferably 50 to 100 microns, more preferably 75 microns from the opposite surface. Therefore, the depth 80 in the instant application is less than the depth of field of the optical system.
- the introduction of a dye 60 that has particular characteristics into the assay chamber of the microiluidic device is yet an additional modification of the invention that is illustrated in FIG. 3 and described below.
- the introduced dye 60 attenuates the effect of background fluorescence 50 emitted by non-specific binding of the fluorescent detector molecules 52 to the luminal surface 14d of the wall 12d opposite to the optically clear wall 12f, but not the specific fluorescence 56 emitted by the fluorescent-labeled target analyte 54 that is specifically bound to the activated luminal surface 14f of the optically clear wall 12f.
- the sample and any unbound material including unbound fluorescent detector molecules are removed from the chamber and the chamber lumen is washed with a volume of wash solution exceeding or equal to the volume of the chamber lumen as described above.
- an attenuating dye 60 as defined above, is introduced into the lumen of the chamber.
- the optimal concentration of the dye is the highest concentration of the dye that meets the following criteria: the dye must remain in solution under all conditions of transportation, storage and use and must not cause chemical or biochemical effects that alter the results of the assay.
- the dye solution volume is approximately equal to the volume of the chamber.
- the attenuating dye may be included with the fluorescent detector molecules or, optionally, in the wash solution that is used to remove unbound fluorescent detector molecules and the sample from the chamber,
- the attenuating dye 60 includes such standard dyes as amaranth, erioglaueme, brilliant green or combinations of various standard dyes.
- Fluorescent labels include fluorescent molecules from common dye families derived from xanthene (e.g. Fluorescein, Texas Red), cyanine, naphthalene, eoumarin, oxadiazoie, pyrene.
- oxazine, acridine, arylmethme, ietrapyrrole and commercial dyes including TOTO-l , YOYO I, Alexa Fluors, Cy family (e.g. Cy2, Cy5, C 7) and many others, as well as fluorescent molecules useful in time-resolved fluorescence such as chelates of the lanthanides, europium, samarium, and terbium.
- Fluorescence 50 from the fluorescent detector molecules 52 that are non- specifically bound to the luminal surface of the chamber, for example, surface 14d, is "masked" by the one or more attenuating dyes 60 that are introduced into the chamber lumen.
- the specific fluorescence 56 of the fluorescent labeled target analyte 54 bound to the luminal surface 14f of the optically clear wall 12f is not masked.
- non-specific fluorescence that is the fluorescence arising from fluorescent detector molecules nor.uspecifically bound to the wall opposite the optically clear wall in particular, the sensitivity and accuracy of the chamber 10 for detecting the target analyte is increased.
- measuring the concentration of the target analyte of interest is accomplished without the obscuring effect caused by the fluorescence 50 of non-specificaUy bound fluorescence detector molecules 52 on the measurement of the concentration of the target analyte reflected by the optical signal.
- the optics of the instrument are arranged to detect fluorescence only from the optically clear wall I2.f or a portion of wall 12f and from the wall 12d opposite to the optically clear wall while not detecting fluorescence that may be emitted from the side walls or any other wall of the chamber 10,
- the optically clear wall 12f of the chamber is 6 mm x 2 mm.
- the optical signal portion 120 of the 6 mm x 2 mm optically clear wall 12f, the center, for example, is utilized for the optical signal. Accordingly, the signal due to non-specific binding of the fluorescent detector molecules on wall surfaces such as the sides of the chamber other than the opposite wall surface 12d is substantially eliminated.
- the outside dimensions of the chamber may be larger than the optically clear area which in turn may be larger than the portion used to make optical measurements.
- Myoglobin is an exemplary target analyte found in a biological specimen that may be detected in the micro fiuidic device according to the invention described above.
- die exemplary chamber is shallow having a depth 80, for example, of about 75 microns.
- a binding partner, a monoclonal antibody, for example, directed to a specific epitope of myoglobm may be used as the binding partner that is applied to the luminal surface 14f of the optically clear wail 12f.
- Another monoclonal antibody directed to a different epitope of myoglobin is labeled with a fluorescent detector molecule such as fluorescent chelates of europium.
- the fluorescently labeled monoclonal antibody is mixed with the biological specimen that may contain the myoglobin target analyte. After sufficient incubation time, the fluorescently labeled monoclonal antibody binds the myoglobin analyte to form a fluorescently labeled myoglobin target analyte.
- non-specific fluorescence from the luminal surface 14d of the opposite wall 12d caused by non-specific binding of the fluorescent detector molecule, and specific fluorescence from the binding of the fluorescently labeled myoglobin target analyte to the specific monoclonal antibody-binding partner on the luminal surface 14f of the optically clear wall 12f, is measured by an optical detector 100.
- the measured optical signal from the assay chamber 10 includes fluorescence 50 from non-specific binding of fluorescent detector molecules 52 to the untreated luminal surface 14d of the wall 12d and fluorescence 56 emitted by the fluorescent chelates of europium labeled myoglobin target analyte 55 specifically bound to the monoclonal antibody binding partner 57 on the luminal surface 1.4f of the optically clear wall 12f, leading to an artificially elevated fluorescence value that does not accurately reflect the concentration of myoglobin in the biological specimen.
- the remaining non-specifically bound fluorescent detector molecules on the luminal surfaces of the chamber particularly on the luminal surface 14d of wall 12d opposite the optically clear wall 12f of the shallow chamber, interfere with the true and specific optical signal emitted from the fluorescently labeled myoglobin target analyte bound to the specific monoclonal antibody binding partners on the luminal surface 14f of the optically clear wall 12f.
- the method to detect the target analyte myoglobin preferably also incorporates the addition of an attenuating dye such as but not limited to amaranth, or combinations of dyes as described above.
- an attenuating dye such as but not limited to amaranth, or combinations of dyes as described above.
- the exemplary chamber is a shallow chamber having a depth 80, for example, of about 75 microns. After sufficient incubation to allow binding to occur, unbound fluorescent labeled monoclonal antibodies directed to the myoglobin target analyte are removed and the chamber lumen 16 is washed with a volume of wash reagent exceeding or equal to the volume of the chamber lumen.
- the wash reagent may contain or may be free of a attenuating dye, amaranth in this example, as described above with respect to FIG. 3. If the wash reagent does not contain the attenuating dye, the dye is added to the chamber lumen after the wash.
- a attenuating dye amaranth in this example, as described above with respect to FIG. 3.
- the wash reagent does not contain the attenuating dye, the dye is added to the chamber lumen after the wash.
- non-specific binding of fluorescent detector molecule to the untreated luminal surface 14d of the assay chamber 10 occurs, as discussed above with respect to FIG. 2.
- the amaranth dye 60 molecules positioned between the non-specifically bound fluorescent, detector molecules 52 on the luminal surfaces 14d of the wall 12d opposite to the optically clear wall 12f, in particular, and the optical system 92 effectively attenuate the non- specific fluorescence.
- amaranth in this example leads to an accurate determination of the specific fluorescence of the myoglobin target analyte 55 bound to the monoclonal antibody-binding partner 57 that is coated on the luminal surface 14f of the optically clear wall 12f.
- the assay chamber 10 is a microfluidic element within a microfluidic assay device, in order to achieve the short incubation times and small sample and reagent volumes tha t are well-known characteristics of microfluidic assay devices. These characteristics can only be achieved if the assay chamber is kept shallow as disclosed abo ve, preferably with depth 10 - 200 microns. If the assay chamber is excessively deep, mass transport by diffusion wil l require long incubation times, and filling and washing of the assay chamber will require larger volumes of costly reagents. However, referring again to FIG.
- a competitive binding assay may be performed.
- a binding partner for the target analyte is coated on the luminal surface of the optically clear wall, as described previously.
- Fluorescently labeled target analyte molecules are prepared that compete with the target analyte for binding specifically to the binding partner coated on the luminal surface of the optically clear wall.
- the fiuorescenfiy labeled target analyte molecules and the unlabeled analyte molecules in the sample compete to bind with the binding partner coated on the luminal surface of the optically clear wal l.
- quantitation of unlabeled analyte in the sample is based on measurement of iluorescence from specifically bound fluorescent molecules on the luminal surface of the optically clear wall. Fluorescence from non-speciflcally bound fluorescent molecules on other surfaces within the depth of field of the optical system degrades the analytical performance of the assay, and the use of an attenuating dye according to the present invention resolves this problem.
- Latex nanoparticles labeled with fluorescent chelates of europium were dispensed directly onto the luminal surface of the wall opposite the optically clear wall of the polystyrene chambers (6 mm x 2,5 mm x ,075mm) described above, ⁇ x 19 5 , 1 x 10° or 1 x 10 ' nanoparticles were added to the surface in 1 uL aqueous buffer and allowed to air dry. An optically clear wall that was not treated was then ultrasonicaily welded onto the chamber to form the assay chamber 10 described above,
- each of the assay chambers described above was then washed three times with 100 uL of an aqueous solution without attenuating dye in order to remove loosely bound material on the luminal surface of the chamber. Fluorescence was measured after each wash using 340 mil excitation and collecting the emitted light using a 615 nm band pass filter. The amount of fluorescence from the luminal surface 14d of the wall 12d opposite the optically clear wall 12f was measured through the optically clear wall 12f, As shown in Table I below, although subsequent washes continued to remove additional fluorescence from the surface, the first three washes removed the majority of the loosely bound nanoparticles, as the fluorescence decreased 92% after the first wash.
- a micro fluidic device having an assay chamber having an assay chamber.
- the assay eh amber has a lumen enclosed by walls and an optional inlet and an outlet port.
- One chamber wall or alternatively, a portion of the chamber wall is optically clear for transmission of excitation and fluorescent light emitted from within the chamber to an optical detector outside the chamber for measuring the amount of fluorescence within the chamber.
- the luminal surface or a portion of the optically clear wall of the chamber is coated (activated) with specific binding partners, as defined above, for a target analyte of interest in the biological sample.
- the luminal surface of the wall opposite the optically clear wall is untreated prior to ini iating an assay.
- the biological sample is mixed with a fluorescent detector molecule that includes another binding partner specific for the target analyte.
- This binding partner may be the same as or, optional ly, different than the binding partner coated on the optically clear surface.
- the sample and the fluorescent detector molecule either individually or in combination are introduced into the lumen of the assay chamber. Alternatively, the sample is added to the chamber, the chamber is washed, followed b adding the fluorescent detector molecule to the chamber.
- the solution including the biological sample and the fluorescent detector molecules are removed from the chamber.
- the chamber is washed with a volume of wash reagent, such as an aqueous buffer, exceeding or equal to the volume of the chamber.
- the chamber is next filled with a volume of a solution such as an aqueous buffer, including one or more attenuating dyes.
- the dye solution volume is
- a wash solution may include the dye.
- the optical signal produced in the lumen of the chamber is measured by an optical detector while the attenuating dye is present in the chamber lumen and the optical signal is compared with a standard curve to determine the concentration of the target analyte in the sample.
Abstract
Description
Claims
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JP2014547297A JP2015500500A (en) | 2011-12-15 | 2012-12-06 | Fluorescence measurement and method for disposable microfluidic devices |
AU2012352725A AU2012352725A1 (en) | 2011-12-15 | 2012-12-06 | Fluorescent measurement in a disposable microfluidic device, and method thereof |
CA2859019A CA2859019A1 (en) | 2011-12-15 | 2012-12-06 | Fluorescent measurement in a disposable microfluidic device, and method thereof |
EP12805550.6A EP2791656A1 (en) | 2011-12-15 | 2012-12-06 | Fluorescent measurement in a disposable microfluidic device, and method thereof |
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US13/326,999 US20120208292A1 (en) | 2010-05-31 | 2011-12-15 | Fluorescent measurement in a disposable microfluidic device, and method thereof |
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WO2011151250A1 (en) * | 2010-05-31 | 2011-12-08 | Boehringer Ingelheim Microparts Gmbh | Method and device for optical examination |
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WO2011151250A1 (en) * | 2010-05-31 | 2011-12-08 | Boehringer Ingelheim Microparts Gmbh | Method and device for optical examination |
US20120208292A1 (en) * | 2010-05-31 | 2012-08-16 | William Lewis | Fluorescent measurement in a disposable microfluidic device, and method thereof |
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CN112113934A (en) * | 2019-06-19 | 2020-12-22 | 泰州医药城国科化物生物医药科技有限公司 | Method for improving cell calcium flow fluorescence detection |
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