EP2417436A1 - Device and method for the verification and quantitative analysis of analytes, particularly mycotoxins - Google Patents

Device and method for the verification and quantitative analysis of analytes, particularly mycotoxins

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Publication number
EP2417436A1
EP2417436A1 EP10712339A EP10712339A EP2417436A1 EP 2417436 A1 EP2417436 A1 EP 2417436A1 EP 10712339 A EP10712339 A EP 10712339A EP 10712339 A EP10712339 A EP 10712339A EP 2417436 A1 EP2417436 A1 EP 2417436A1
Authority
EP
European Patent Office
Prior art keywords
analytes
cartridge
immunoassay
probes
analyte
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10712339A
Other languages
German (de)
French (fr)
Inventor
Jens Burmeister
Ingmar Dorn
Viktoria Bazilyanska
Ulrich Raczek
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Intellectual Property GmbH
Original Assignee
Bayer CropScience AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer CropScience AG filed Critical Bayer CropScience AG
Priority to EP10712339A priority Critical patent/EP2417436A1/en
Publication of EP2417436A1 publication Critical patent/EP2417436A1/en
Withdrawn legal-status Critical Current

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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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G01N33/5304—Reaction vessels, e.g. agglutination plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
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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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N21/64—Fluorescence; Phosphorescence
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    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
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    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • 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
    • 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
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • 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
    • G01N2021/0328—Arrangement of two or more cells having different functions for the measurement of reactions
    • 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
    • G01N2021/0346—Capillary cells; Microcells
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • 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
    • G01N21/0332—Cuvette constructions with temperature control
    • 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
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/274—Calibration, base line adjustment, drift correction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/02—Food
    • G—PHYSICS
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    • G01N33/02—Food
    • G01N33/10—Starch-containing substances, e.g. dough

Definitions

  • the present invention relates to an apparatus and a method for the detection and quantitative analysis of analytes and their use for the detection and quantitative analysis of mycotoxins.
  • analyzes are often based on the demonstration of an interaction between a molecule that is present in a known amount and position (the molecular probe) and an unknown molecule to be detected (the molecular target or target molecule).
  • a probe which is usually fixed to a carrier, and with a target molecule in a sample solution is brought into contact and incubated under defined conditions.
  • a specific interaction takes place between probe and target, which can be detected in various ways.
  • the detection is based on the fact that a target molecule only binds specifically to certain probe molecules. The binding is much more stable than the binding of target molecules to probes that are not specific for the target molecule.
  • the target molecules that have not been specifically bound can be removed by washing while the specifically bound target molecules are captured by the probes.
  • the detection of the specific interaction between a target and its probe can then be carried out on a so-called marker by a variety of methods, which usually depend on the type of marker, which introduced before, during or after the interaction of the target molecule with the probes has been.
  • markers are fluorescent groups, so that specific target-probe interactions with high spatial resolution and compared to other conventional detection methods, especially mass-sensitive methods, can be read out fluorescently optically with little effort (A. Marshall, J Hodgson, DNA Chips: An array of possibilities, Nature Biotechnology 1998, 16, 27-31, G. Ramsay, DNA Chips: State of the Art, Nature Biotechnology 1998, 16, 40-44).
  • An evanescent field biochip comprises an optical waveguide capable of detecting changes in the optical properties of a medium adjacent to the waveguiding layer. If light is transported as a guided mode in the waveguiding layer, the light field at the interface medium / waveguide does not drop abruptly, but decays exponentially in the so-called detection medium adjoining the waveguide. This exponentially decaying light field is called an evanescent field. If the optical properties of the medium adjacent to the waveguide change within the evanescent field, this can be detected by means of a suitable measurement setup. Thus, the detection of the specific binding of target molecules to probes immobilized on the waveguide can take place via the changing optical properties of the waveguide / immobilizate boundary layer.
  • a fluorescence signal is detected in the evanescent field.
  • the fluorescently labeled binding pair probe / target molecule is excited by an evanescent field.
  • An example of an evanescent field biochip is given in US 5,959,292.
  • the prior art in particular uses the dry-type assay technology, in which all reagents in the dry state in the cassette are optionally available in separate chambers.
  • the sample liquid is usually conveyed by means of microfluidic channels from one chamber to the next.
  • WO 2005/088300 describes, for example, an integrated microfluidic cartridge for blood analysis, which consists of a lower and an upper part of the body. Both elements are structured with chambers and channels, which are closed by the joining of the two parts.
  • the test cassette has one or more pre-treatment elements (pre-treatment chamber) for preparing a sample, one or more multi-layered dry assay elements (detection chamber) for detecting one or more target molecules of a sample liquid, and one or more channels (average ⁇ 3 mm), which connect the pretreatment elements to the multilayer dry assay elements.
  • the pretreatment elements are, in particular, filter elements or elements with porous properties in the form of a channel or a (micro / nano) cushion, which optionally carry dry reagents.
  • the sample is first passed through the pretreatment elements, then into the multilayer dry assay element.
  • the multilayer dry assay detection element comprises at least one functional layer carrying probes for a qualitative and quantitative assay of the target molecules in dry and stable form.
  • This reagent layer consists of a water-absorbing layer in which stimulable probes are reasonably regularly distributed in a hydrophilic polymer binder material (gelatin, agarose, etc.).
  • the detection is carried out by reflection photometry through a light-transparent window, by irradiation of a detection layer in the multilayer dry assay element in which the optically excitable liquid has diffused from the detection reaction. Capillary forces or pressure are used to carry the sample.
  • this device Disadvantage of this device is that the structure of the multilayer dry assay element is complex and the mixing of the analyte is not optimal with the detection reagents. In addition, an exact time control of the individual reaction steps, in particular the volume and incubation times is not possible, so that the test results are quantitatively not reproducible. Referencing is not described.
  • LFA Lateral Flow Assay
  • LFA Lateral flow assays
  • a direct, competitive immunoassay can be carried out on a nitrocellulose strip, wherein the sample to be analyzed is pulled through the entire nitrocellulose strip due to capillary forces.
  • the zone in which the anti-analyte antibody was immobilized serves as a detection zone for the streak test.
  • LFA assay for the detection of mycotoxins (e.g., deoxynivalenol) is the "Reveal Assay” (test cassette) from Neogen, Lansing, MI, USA with the associated "AccuScan” reader.
  • the cartridge is inserted into the reader and the device takes a picture of the result range of the strip test.
  • the reader interprets the result image and, if a line has been detected, a rating is given.
  • the device eliminates the subjectivity of the interpretation and gives an objective, traceable documentation of the test result.
  • the test described is simple and relatively fast to carry out and does not require elaborate read-out devices. The disadvantage is that the method only allows qualitative or semiquantitative mycotoxin detection.
  • WO 2007/079893 describes a method for rapid detection of mycotoxin in which a supported substance library of immobilized binding partners for mycotoxins and / or probes for mycotoxins in spatially separated measurement areas is applied to the surface of a thin-film waveguide, a mycotoxin and probes containing this mycotoxin sample in Contact with the immobilized binding partners is brought and the reaction of the immobilized binding partner with the mycotoxins and / or recognition elements of mycotoxins is detected by a signal change in the evanescent field, that is at the interface to the waveguide.
  • measuring ranges for referencing the same chemical or Optical parameters for example the intensity of the locally available excitation light
  • Optical parameters for example the intensity of the locally available excitation light
  • the number and position of the measuring ranges for referencing in the above-mentioned arrangement of measuring ranges is arbitrary.
  • EP-A 0093 613 describes a method for calibrating an assay for quantifying a target molecule in a sample liquid by means of a sensor based on fluorescence excitation in the evanescent field of an optical waveguide having a first measurement range (measurement range) for the specific binding of a first label Label in an amount dependent on the presence of an analyte in the sample, and a second measurement range (calibration range) for binding a second label, wherein the binding of the second label does not affect the presence of the analyte in the sample becomes.
  • Different binding pairs are used in the measurement ranges and calibration ranges, but they are of similar nature.
  • the quantity of the second label in the calibration area during the assay gives a signal value for a predefined concentration of the analyte within a concentration range.
  • Both measuring ranges are placed close to each other, on the same basic structure, in order to minimize differences caused by possible local variations of the sensor.
  • the signal value of the measurement range is divided by the signal value of the calibration range placed close to each other to correct the non-specific effects of the sensor on the signal.
  • the structure of the sensor and the direction of the excitation beam are not defined.
  • WO 2004/023142 describes a method for calibrating an assay for quantifying a target molecule in a sample liquid by means of a sensor based on fluorescence excitation in the evanescent field of an optical waveguide, on the recognition elements and reference molecules (Cy5-BSA, bovine serum albumin) in separated parallel alternating microarrays orthogonal to the propagation direction of guided in the Evaneszenzfeld sensor platform excitation light in Messspots or reference spots are spotted.
  • the net signal intensity of the measurement spot is divided by the average of the net signal intensities of the adjacent reference spots of the same row arranged in the propagation direction of the excitation light. This referencing compensates for the local differences in available excitation light intensity orthogonal to the light propagation direction both within each microarray and between different microarrays.
  • a microfluidic cartridge for the qualitative and / or quantitative analysis of analytes, in particular of mycotoxins, which contains all the reagents required for carrying out the test method in dry form.
  • the cartridge according to the invention has a structured body into which cavities have been introduced, which are connected to one another by channels.
  • the cartridge has at least one inlet for introducing a sample liquid containing mycotoxins, at least one reagent chamber, and at least one detection chamber.
  • One or more labeled mycotoxin probes for reaction with the mycotoxins from the sample fluid and labeled homing probes for reaction with a referencing antigen are housed in the reagent chamber in dry form.
  • the bottom of the detection chamber consists of a thin-film waveguide (PWG biochip) comprising a first optically transparent layer (a) on a second optically transparent layer (b) having a lower refractive index than layer (a) and in which an optical grating is introduced, wherein the grating is oriented perpendicular to the path of an excitation light, which is coupled by means of the optical grating in the thin-film waveguide.
  • PWG biochip thin-film waveguide
  • detection reagents are immobilized; Namely, in series of spatially separated measurement areas, a mycotoxin assay (immunoassay) in the form of a substance library of immobilized binding partners for mycotoxins and / or mycotoxin probes and an independent control assay comprising an immobilized homing antigen are applied.
  • the arrays are applied to the PWG biochip in such a way that the measurement areas are aligned in rows parallel to the optical grid. In the direction of the excitation light above and below each series of immunoassay is a series of the control assay (see FIG.
  • the first object of the present invention is therefore a cartridge for the detection and quantitative analysis of analytes in a sample liquid, comprising a structured body, are introduced into the cavities, which are connected to each other through channels, said cartridge containing at least one inlet for introducing the analytes Probe Letkeit, at least one reagent chamber and at least one detection chamber, wherein - - a. in the reagent chamber one or more labeled analyte probes for reaction with the analytes from the sample liquid and one or more labeled homing probes for reaction with a referencing antigen in dry form are accommodated,
  • the bottom of the detection chamber is a thin-film waveguide comprising a first optically transparent layer (a) on a second optically transparent layer (b) having a lower refractive index than layer (a), wherein in layer (a) or (b ) an optical grating is introduced, which is oriented perpendicular to the path of an excitation light, which is coupled by means of the optical grating in the thin-film waveguide,
  • an immunoassay in the form of a substance library of immobilized binding partners for analytes and / or for analyte probes in rows of spatially separated measurement areas and an independent control assay comprising the referencing antigen immobilized in rows of spatially separated measurement areas are applied and
  • the respective rows are oriented parallel to the optical grating and in the direction of the excitation light above and below each row of the immunoassay a series of
  • control assay is selected such that the referencing antigen has a molecular weight similar to the analyte, and the homing probe has similar binding properties as the analyte probes (affinity, binding kinetics).
  • the control assay must also show no cross-reactivity to the immunoassays and the antigen must not naturally occur in the matrix examined.
  • the analytes are mycotoxins.
  • an immunoassay as described in WO 2007/079893 is used, the content of which is integrated by reference.
  • Preferred immunoassay sequences of mycotoxin-protein conjugates e.g. Mycotoxin-BSA conjugates.
  • control assays are assays against mycotoxins that do not naturally occur in the matrix examined.
  • the control assay is preferably selected to detect a molecule ⁇ 1000 g / mol.
  • a control assay for fluorescein and a number of control protein conjugates, eg fluorescein-BSA are applied to the PWG biochip.
  • the PWG biochip for example, consists of a glass carrier which is coated with a layer of tantalum pentoxide.
  • the layer thickness is 40 to 160 nm, preferably 80 to 160 nm, particularly preferably 120 to 160 nm, very particularly preferably 155 nm.
  • the glass substrate contains an optical grating with a grating depth of 3 to 60 nm, preferably 5 to 30 nm, particularly preferably 10 to 25 nm, very particularly preferably 18 nm and a grating period of 200 to 1000 nm, preferably 220 to 500 nm, particularly preferably 318 nm.
  • the grating has a single period, that is to say it is monodiffractive.
  • the tantalum pentoxide surface is usually coated with dodecyl phosphate in the form of a monolayer.
  • Analyte-protein conjugates preferably mycotoxin-BSA conjugates and homing antigen-protein conjugates, preferably fluorescein-BSA conjugates, are immobilized on this surface.
  • the protein conjugates are usually used in concentrations of 0.1 to 5 mg / ml, preferably 0.2 to 2 mg / ml, more preferably 0.5 to 1.5 mg / ml, most preferably 1 mg / ml applied to the surface and adsorbed there.
  • one or more methods selected from the group consisting of ink jet spotting, mechanical spotting by pen or pen, microcontact printing, fluidic contacting of the measurement regions with the biological or biochemical or synthetic recognition elements may be used their supply in parallel or crossed microchannels, under the effect of pressure differences or electrical or electromagnetic potentials.
  • the still free areas of the PWG chip surface are passivated by treatment with BSA in order to suppress non-specific binding.
  • the PWG biochip represents the bottom of the detection chamber of the cartridge according to the invention and is integrated into the cartridge.
  • the cartridge consists of a structured body, are introduced into the chambers and channels, the chambers are preferably introduced into the body so that they are formed on at least one side by the application of a closure unit.
  • the structured body is closed at the top and bottom by means of a closure unit with the exception of the inlet, the bottom of the detection chamber and optional ventilation openings.
  • the biochip is positioned in front of the closure unit and held in position by the closure unit.
  • the closure unit is preferably a closure film.
  • a precisely defined volume of sample liquid is conveyed in the channels and in the chambers, which is made possible by the design of the channels and the chamber and the use of a suitable means for transporting the sample liquid. Reaction times can also be precisely controlled, which contributes to better reproducibility of the analysis.
  • the proper design of the chamber and channels ensures an optimal flow profile with reduced dead volume and possibly optimal contact with the immobilized detection reagents.
  • the channels connect the inlet, the reagent chamber and the detection chamber with each other and usually have a diameter of 0.1 to 2.5 mm, preferably 0.5 to 1.5 mm, particularly preferably 1 mm.
  • the reagent chamber has a reagent pad, on which the analyte and Referenzierungsonden esp. Antibodies for mycotoxins and fluorescein are housed.
  • the reagent pad is selected to meet the requirements of the detection chamber with respect to the required liquid volume of the supernatant solution and the concentration of the individual components in this solution.
  • the reagent pad is usually made of a fibrous or porous material, e.g. fine particles or tissue into which reagents (adsorbed on, fixed on, dispersed in, dried in) were placed.
  • a preferred reagent pad is made of glass or polymers, e.g. Cellulose.
  • Reagent pads are used, which are also used in so-called lateral flow assays and commercially available in various forms.
  • a preferred reagent chamber requires a liquid volume of 10 to 100 .mu.l, preferably 20 to 60 .mu.l, more preferably 40 .mu.l and analyte and Referenzierungsonden dissolved therein in a concentration of 10 "7 M to 10 " 10 M, preferably nanomolar concentrations.
  • the reagent pad is selected, preferably from extra thick glass filters from Paill Corporation (pore size 1 ⁇ m, typical thickness 1270 ⁇ m (50 mils), typical water flow rate 210 mL / min / cm 2 at 30 kPa), wherein two circular filter pieces with a suitable diameter (usually from 5 to 10 mm) are stacked on top of each other.
  • the resulting reagent pad is usually impregnated with about 100 ⁇ l of the solution containing the fluorescently labeled probes and usually other components to aid in impregnation. The impregnation is carried out, for example, by drying or lyophilization.
  • the reagent pad is usually operated in the cartridge so that it is wetted with about 80 ⁇ l of sample fluid (e.g., mycotoxin extract).
  • sample fluid e.g., mycotoxin extract
  • Another object of the present invention is a method for the detection of analytes, in particular mycotoxins, by means of the cartridge according to the invention.
  • Second object of the present invention is a method for the quantitative analysis of analytes, comprising the following steps:
  • the mycotoxins are present in a solid matrix, they are usually comminuted in an optional first step of the process according to the invention, after which the mycotoxins are extracted from the matrix with a suitable solvent.
  • suitable solvent examples include extractants are aqueous solutions of methanol, ethanol or acetonitrile.
  • solid matrices are wheat, corn, barley, rye, peanuts, hazelnuts, etc. If the extract contains more than 10% of the nonaqueous solvent, a dilution step is usually required before filling the cartridge. Liquid matrices (milk, fruit juice, wine etc.) can be filled into the cartridge directly or after a suitable dilution.
  • the user fills the extract or the sample solution in the cartridge and closes the cartridge.
  • the cartridge is then inserted into a reader.
  • the reader includes a pump that pumps air into the cartridge, transporting the solution from the sample inlet into the reaction chamber where it wets the reagent pad applied there.
  • the antibodies When wetting the reagent pad, the antibodies are removed from the reagent pad using the extract and mixed with the extract.
  • the incubation time of the extract in the reagent pad is preferably 1 to 20 minutes, more preferably 3 to 7 minutes.
  • the pump now again pumps air into the cartridge and thereby moves the liquid volume - into the detection chamber above the PWG biochip. Again, there is an incubation step, which usually takes 1 to 100 minutes, preferably 5 to 15 minutes.
  • the cartridge over the duration of the process to a temperature which is preferably 20 to 37 0 C, more preferably 25 0 C, tempered.
  • a laser beam is coupled into the optical grating.
  • the extensive illumination of the PWG biochip stimulates the labeled antibodies to fluoresce.
  • a camera and a suitable fluorescence filter the fluorescence image of the biochip is recorded.
  • An image-evaluating software which is installed in the computer of the reader, now determines the fluorescence intensity of the mycotoxin and control assay ranges. By dividing the fluorescence intensity of the mycotoxin assay measurement range by the mean of the fluorescence intensities of the control assay measurement regions adjacent in the direction of the excitation light, a referenced fluorescence intensity of the mycotoxin assay measurement region is obtained.
  • the quantitative relationship between the referenced fluorescence intensities of the mycotoxin assay ranges and the concentration of a mycotoxin in the solution pipetted into the cartridge is usually determined by taking calibration curves.
  • the resulting mathematical relationships are stored on the computer of the reader.
  • the referenced fluorescence intensity is determined after taking the fluorescence image and the corresponding mycotoxin concentration is calculated by reference to the calibration curve.
  • the mycotoxin value is displayed on the screen of the reader.
  • Fig. 1 Construction of the mycotoxin array
  • Fig. 2 Structure of the cartridge
  • Fig. 3 PWG biochip side view
  • Fig. 4 Dimensions of the PWG biochip.
  • Waveguiding layer 10 Monolayer of dodecyl phosphate / adhesion promoting layer
  • the cartridge (1) consists of a structured body, were introduced into the channels and cavities.
  • the cartridge according to the invention was produced in an injection molding process.
  • the body consists of a black polyoxymethylene (POM) plate in which the channels and chambers have been drilled and milled off.
  • POM polyoxymethylene
  • the cartridge (1) comprises an inlet (2) for introducing a Probenfiüsstechnik with the analyte to be detected in a sample chamber of the cartridge (1), a reagent chamber with a reagent Päd (4), in which the sample liquid via a channel (3) and a detection chamber (5) into which the sample liquid is conveyed via a further channel (3) and which comprises a PWG biochip (6).
  • Both the PWG biochip (6) and the reagent pad were held between two polyolefin sheets, in the POM plate, which also served as sealing sheets to seal the test cassette.
  • the top closure film was 180 ⁇ m thick and the bottom closure film was 80 ⁇ m thick.
  • the bottom foil had a window in the area of the PWG biochip (6) which allowed free access to the measurement region of the PWG biochip (6).
  • the sample liquid was introduced into the sample chamber through the inlet (2) at the start of the test, and the inlet (2) was sealed airtight with a suitable lid. With the aid of the transport unit, a defined volume of air was introduced into the cartridge (1) at the inlet. This volume of air displaced the sample fluid so that it flowed into the reagent chamber (4) and completely wetted the reagent pad.
  • the antibodies were dissolved, mixed with the sample liquid, and bound with the mycotoxins contained in the sample liquid (mycotoxin-antibody conjugate).
  • the free binding sites of the antibodies were increasingly saturated with increasing amount of mycotoxins in the sample liquid.
  • the sample containing liquid was mycotoxin Antikö ⁇ er conjugates and conveys the antibody for fluorescein in a next step in the detection chamber (5).
  • the detection chamber (5) was completely filled with the sample liquid.
  • the duct system was completely vented. The venting of the complete channel system took place via venting holes applied in the upper closure panel.
  • the detection chamber (5) comprised a PWG biochip (6).
  • the PWG biochip (6) is shown schematically in plan view in FIG. 2 and schematically in side view in FIG.
  • the PWG biochip (6) in the detection chamber (5) consisted of a 10 mm x 12 mm glass plate (8) with a thickness of 0.7 mm (12.0 +/- 0.05 mm x 10.0 +/- 0.05 mm x 0.70 +/- 0.05 mm).
  • On one side of the PWG chip (6) was a 155 nm thin waveguiding layer (9) of Ta 2 O 5 (tantalum pentoxide).
  • the measuring region of the chip consisted of a central 10 mm x 6 mm square area. Parallel to this measurement region is a 500 ⁇ m wide crescent-shaped band: the grating (7) for coupling the excitation beam.
  • the positional accuracy of the grating (7) to the edges was +/- 0.05 mm.
  • the grating depth was 18 nm and the grating 318 nm with a duty cycle of 0.5.
  • the mycotoxic antibody conjugate and optionally antibodies with free binding sites and the antibodies for the fluorescein pass to the immunoassay (12) from immobilized analyte-BSA conjugates or the control assay (11, 13) on the PWG biochip (6).
  • Antibodies with free binding sites entered into specific binding with the corresponding immobilized analyte-BSA conjugates.
  • the antibodies saturated with analytes in the sample liquid remained in the solution.
  • the antibodies bound to the immobilized analyte-BSA conjugates and labeled with a fluorescent dye could be excited to fluoresce in the evanescent field of the waveguide.
  • the antibodies in solution and labeled with a fluorescent dye were not excited in this case. In this way, an indirect quantification of the mycotoxins contained in the sample liquid was achieved.
  • the spots were applied to the PWG biochip in alternating rows of 16 BSA-FITC conjugate spots and BSA-DON conjugate spots, respectively, so that the rows ran parallel to the optical grating.
  • the spots were allowed to dry and then treated with the mist of an aqueous BSA solution.
  • the PWG biochips were washed and then allowed to dry.
  • the PWG biochips were glued into cartridges with double-sided adhesive tape.
  • the cartridges contained a sample chamber for sample collection, a reagent chamber with a glass fiber pad and a detection chamber for the PWG biochip. The chambers were connected by channels.
  • the glass fiber material was impregnated with solutions of nanomolar concentrations of antibodies labeled with the fluorescent dye DY-647 (Dyomics, Germany) using monoclonal antibodies against deoxynivalenol and fluorescein.
  • PBS phosphate buffered saline
  • ovalbumin 0.1% ovalbumin
  • Tween 0.5% sucrose
  • the fluorescence intensities obtained for each DON spot were divided by the average of the fluorescence intensities of the BSA-FITC spot located above and below the respective DON spot. The mean values of the thus referenced fluorescence intensities of all 16 DON spots were determined. The resulting concentration-dependent, referenced fluorescence intensities were adjusted by a sigmoidal fit using the computer program Origin 7G (Origin Lab Corporation, USA).
  • the homogenized sample contained 888 mg / kg (ppb) of DON.
  • 5 g of the flour sample was extracted with 25 ml of 70% methanol by vigorous shaking for 3 min. The extract was allowed to settle and the supernatant was diluted 1: 3 with buffer. The diluted extract was filled in 7 different cartridges. The cartridges were then measured in the read-out device MyToLab as described above and the referenced fluorescence intensities of the DON spots were determined. In relation to the standard curve described above, the concentrations of DON in ppb were determined to obtain values of 1042, 757, 710, 660, 431, 728 and 984 ppb. The mean DON determination was 760 ppb with a percent standard deviation of 27%.

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Abstract

The present invention relates to a device and a method for the verification and quantitative analysis of analytes and their application for the verification and quantitative analysis of mycotoxins.

Description

Vorrichtung und Verfahren zum Nachweis und zur quantitativen Analyse von Analyten insbesondere Mykotoxinen Device and method for the detection and quantitative analysis of analytes, in particular mycotoxins
Die vorliegende Erfindung betrifft eine Vorrichtung und ein Verfahren zum Nachweis und zur quantitativen Analyse von Analyten und ihre Verwendung zum Nachweis und zur quantitativen Analyse von Mykotoxinen.The present invention relates to an apparatus and a method for the detection and quantitative analysis of analytes and their use for the detection and quantitative analysis of mycotoxins.
In der Biochemie und Medizin basieren Analysen häufig auf dem Nachweis einer Wechselwirkung zwischen einem Molekül, das in bekannter Menge und Position vorhanden ist (der molekularen Sonde) und einem nachzuweisenden, unbekannten Molekül (dem molekularen Ziel- oder Targetmolekül).In biochemistry and medicine, analyzes are often based on the demonstration of an interaction between a molecule that is present in a known amount and position (the molecular probe) and an unknown molecule to be detected (the molecular target or target molecule).
Zum Nachweis einer Wechselwirkung wird eine Sonde, die üblicherweise an einem Träger fixiert ist, und mit einem in einer Probelösung befindlichen Targetmolekül in Kontakt gebracht und unter definierten Bedingun- gen inkubiert. Infolge der Inkubation findet zwischen Sonde und Target eine spezifische Wechselwirkung statt, die auf verschiedene Weise detektiert werden kann. Der Nachweis basiert auf der Tatsache, dass ein Targetmolekül nur mit bestimmten Sondenmolekülen eine spezifische Bindung eingeht. Die Bindung ist deutlich stabiler als die Bindung von Targetmolekülen an Sonden, die für das Targetmolekül nicht spezifisch sind. Die Targetmoleküle, die nicht spezifisch gebunden worden sind, können durch Waschung entfernt werden, während die spezifisch gebundenen Targetmoleküle durch die Sonden festgehalten werden.To detect an interaction, a probe, which is usually fixed to a carrier, and with a target molecule in a sample solution is brought into contact and incubated under defined conditions. As a result of the incubation, a specific interaction takes place between probe and target, which can be detected in various ways. The detection is based on the fact that a target molecule only binds specifically to certain probe molecules. The binding is much more stable than the binding of target molecules to probes that are not specific for the target molecule. The target molecules that have not been specifically bound can be removed by washing while the specifically bound target molecules are captured by the probes.
Bei modernen Tests sind eine Vielzahl an Sonden in Form einer Substanzbibliothek als Matrix (Array) auf einem Träger abgelegt, so dass eine Probe parallel an mehreren Sonden gleichzeitig analysiert werden kann (D. J. Lockhart, E. A. Winzeler, Genomics, gene expression and DNA arrays; Nature 2000, 405, 827-836).In modern tests, a large number of probes in the form of a substance library are stored as a matrix (array) on a support so that a sample can be analyzed simultaneously on several probes simultaneously (DJ Lockhart, EA Winzeler, Genomics, Gene expression and DNA arrays; 2000, 405, 827-836).
Der Nachweis der spezifischen Wechselwirkung zwischen einem Target und seiner Sonde kann dann anhand eines so genannten Markers durch eine Vielzahl von Verfahren erfolgen, die in der Regel von der Art des Markers abhängen, der vor, während oder nach der Wechselwirkung des Targetmoleküls mit den Sonden eingebracht worden ist. Typischerweise handelt es sich bei solchen Markern um fluoreszierende Gruppen, so dass spezifische Target-Sonden- Wechselwirkungen mit hoher Ortsauflösung und im Vergleich zu anderen herkömmlichen Nachweismethoden, vor allem massensensitiven Methoden, mit geringem Aufwand fluores- zenzoptisch ausgelesen werden können (A. Marshall, J. Hodgson, DNA Chips: An array of possibilities, Nature Biotechnology 1998, 16, 27-31; G. Ramsay, DNA Chips: State of the art, Nature Biotechnology 1998, 16, 40-44).The detection of the specific interaction between a target and its probe can then be carried out on a so-called marker by a variety of methods, which usually depend on the type of marker, which introduced before, during or after the interaction of the target molecule with the probes has been. Typically, such markers are fluorescent groups, so that specific target-probe interactions with high spatial resolution and compared to other conventional detection methods, especially mass-sensitive methods, can be read out fluorescently optically with little effort (A. Marshall, J Hodgson, DNA Chips: An array of possibilities, Nature Biotechnology 1998, 16, 27-31, G. Ramsay, DNA Chips: State of the Art, Nature Biotechnology 1998, 16, 40-44).
Besonders vorteilhaft ist in diesem Zusammenhang die Verwendung eines Evaneszent-Feld-Biochips als Träger für die Sondenmoleküle. Ein Evaneszent-Feld-Biochip umfasst einen optischen Wellenleiter, mit dem Änderungen der optischen Eigenschaften eines Mediums detektiert werden können, das an die wellenleitende Schicht grenzt. Wird Licht als geführte Mode in der wellenleitenden Schicht transportiert, fällt das Lichtfeld an der Grenzfläche Medium/Wellenleiter nicht abrupt ab, sondern klingt in dem an den Wellenleiter angrenzenden so genannten Detektionsmedium exponentiell ab. Dieses exponentiell abfallende Lichtfeld wird als evaneszentes Feld bezeichnet. Ändern sich die optischen Eigenschaften des an den Wellenleiter grenzenden Mediums innerhalb des evaneszenten Feldes, kann dies über einen geeigneten Messaufbau detektiert werden. Somit kann der Nachweis über die spezifische Bindung von Targetmolekülen an auf dem Wellenleiter immo- biliserten Sonden über die sich ändernden optischen Eigenschaften der Grenzschicht Wellenleiter/Immobilisat erfolgen.Particularly advantageous in this context is the use of an evanescent field biochip as a carrier for the probe molecules. An evanescent field biochip comprises an optical waveguide capable of detecting changes in the optical properties of a medium adjacent to the waveguiding layer. If light is transported as a guided mode in the waveguiding layer, the light field at the interface medium / waveguide does not drop abruptly, but decays exponentially in the so-called detection medium adjoining the waveguide. This exponentially decaying light field is called an evanescent field. If the optical properties of the medium adjacent to the waveguide change within the evanescent field, this can be detected by means of a suitable measurement setup. Thus, the detection of the specific binding of target molecules to probes immobilized on the waveguide can take place via the changing optical properties of the waveguide / immobilizate boundary layer.
Vorzugsweise detektiert man im Evaneszentfeld ein Fluoreszenzsignal. Das fluoreszent markierte Bindungs- paar Sonde/Targetmolekül wird durch ein evaneszentes Feld angeregt. Ein Beispiel eines Evaneszent-Feld- Biochips ist in US 5,959,292 gegeben.Preferably, a fluorescence signal is detected in the evanescent field. The fluorescently labeled binding pair probe / target molecule is excited by an evanescent field. An example of an evanescent field biochip is given in US 5,959,292.
Abhängig von einer auf dem Träger immobilisierten Substanzbibliothek von Sonden und der chemischen Natur der Targetmoleküle können anhand dieses Testprinzips z.B. Wechselwirkungen zwischen Nukleinsäuren und Nukleinsäuren, zwischen Proteinen und Proteinen, Antikörpern und Antigenen sowie zwischen Nuklein- säuren und Proteinen untersucht werden.Depending on a substance library of probes immobilized on the support and the chemical nature of the target molecules, it is possible, on the basis of this test principle, to use e.g. Interactions between nucleic acids and nucleic acids, between proteins and proteins, antibodies and antigens as well as between nucleic acids and proteins are investigated.
Um ein praktisches Schnell-Nachweisverfahren zu ermöglichen, wird schon seit einigen Jahren versucht, Chemo- bzw. Biosensoren-Geräte zu verkleinern und möglichst alle Reagenzien, die zur qualitativen und/oder quantitativen Bestimmung einer Probe benötigt werden, in einer sogenannten Kartusche „ready-to-use" fertig zu stellen. Insbesondere wird die Mikrofluidik-Technologie eingesetzt und dabei angestrebt, kostengünstige, lagerfähige, und einfach zu bedienende Einweg-Kassetten zur Verfügung zu stellen, die zeitecht reproduzierbare Ergebnisse liefern können.In order to enable a practical rapid detection method, it has been attempted for some years to reduce the size of chemo- or biosensor devices and, if possible, to use all reagents required for the qualitative and / or quantitative determination of a sample in a so-called cartridge. In particular, microfluidics technology is being used, with the aim of providing cost-effective, storable, and easy-to-use disposable cassettes that can deliver timely, reproducible results.
Zur Lagerfähigkeit und Transportierbarkeit von Kartuschen wird im Stand der Technik insbesondere die Tro- cken-Assay-Technologie eingesetzt, bei der alle Reagenzien im trockenen Zustand in der Kassette ggf. in getrennten Kammern bereit stehen. Die Probeflüssigkeit wird üblicherweise mittels mikrofluidischen Kanälen von einer Kammer in die nächste weiterbefördert.For the shelf life and transportability of cartridges, the prior art in particular uses the dry-type assay technology, in which all reagents in the dry state in the cassette are optionally available in separate chambers. The sample liquid is usually conveyed by means of microfluidic channels from one chamber to the next.
WO 2005/088300 beschreibt z.B. eine integrierte mikrofluidische Kartusche zur Blutanalyse, die aus einem unteren und einem oberen Körperteil besteht. Beide Elemente werden mit Kammern und Kanälen strukturiert, die durch das Zusammenfügen der beiden Teile geschlossen werden. Die Testkassette weist ein oder mehrere Vorbehandlungselemente (Vorbehandlungskammer) zur Vorbereitung einer Probe, eine oder mehrere mehr- schichtige Trocken-Assay-Elemente (Detektionskammer) zur Erkennung einer oder mehrerer Targetmoleküle einer Probeflüssigkeit, und ein oder mehrere Kanäle (Durchschnitt < 3 mm), die die Vorbehandlungselemente mit den mehrschichtigen Trocken-Assay-Elementen verbinden, auf. Die Vorbehandlungselemente sind insbesondere Filterelemente oder Elemente mit porösen Eigenschaften in Form eines Kanals oder eines (Mik- ro/Nano)-Kissens, die ggf. trockene Reagenzien tragen. Die Probe wird zuerst durch die Vorbehandlungsele- mente, dann in das mehrschichtige Trocken-Assay-Element geführt. Das mehrschichtige Trocken-Assay- Erkennungselement weist mindestens eine funktionelle Schicht, die Sonden für einen qualitativen und quantitativen Assay der Targetmoleküle in trockener und stabiler Form trägt, auf. Diese Reagenzschicht besteht aus einer wasserabsorbierenden Schicht, in der anregbare Sonden einigermaßen regelmäßig in einem hydrophilen Polymerbindematerial (Gelatin, Agarose, usw.) verteilt sind. Die Detektion erfolgt durch Reflektionsphoto- metrie durch ein Licht-durchsichtiges Fenster, durch Einstrahlen einer Detektionsschicht im mehrschichtigen Trocken-Assay-Element, in der die optisch anregbare Flüssigkeit aus der Erkennungsreaktion diffundiert ist. Zur Beförderung der Probe werden kapillarische Kräfte oder Druck eingesetzt. Nachteil dieser Vorrichtung ist, dass der Aufbau des mehrschichtigen Trocken-Assay-Elements aufwändig ist und das Mischen des Analy- ten mit dem Detektionsreagenzien nicht optimal ist. Eine genaue zeitliche Kontrolle der einzelnen Reaktions- schritte, insbesondere der Volumen und Inkubationzeiten ist außerdem nicht möglich, so dass die Testergeb- nisse quantitativ nicht reproduzierbar sind. Eine Referenzierung wird nicht beschrieben.WO 2005/088300 describes, for example, an integrated microfluidic cartridge for blood analysis, which consists of a lower and an upper part of the body. Both elements are structured with chambers and channels, which are closed by the joining of the two parts. The test cassette has one or more pre-treatment elements (pre-treatment chamber) for preparing a sample, one or more multi-layered dry assay elements (detection chamber) for detecting one or more target molecules of a sample liquid, and one or more channels (average <3 mm), which connect the pretreatment elements to the multilayer dry assay elements. The pretreatment elements are, in particular, filter elements or elements with porous properties in the form of a channel or a (micro / nano) cushion, which optionally carry dry reagents. The sample is first passed through the pretreatment elements, then into the multilayer dry assay element. The multilayer dry assay detection element comprises at least one functional layer carrying probes for a qualitative and quantitative assay of the target molecules in dry and stable form. This reagent layer consists of a water-absorbing layer in which stimulable probes are reasonably regularly distributed in a hydrophilic polymer binder material (gelatin, agarose, etc.). The detection is carried out by reflection photometry through a light-transparent window, by irradiation of a detection layer in the multilayer dry assay element in which the optically excitable liquid has diffused from the detection reaction. Capillary forces or pressure are used to carry the sample. Disadvantage of this device is that the structure of the multilayer dry assay element is complex and the mixing of the analyte is not optimal with the detection reagents. In addition, an exact time control of the individual reaction steps, in particular the volume and incubation times is not possible, so that the test results are quantitatively not reproducible. Referencing is not described.
Die Lateral Flow Assay (LFA) Technologie ist auch schon seit vielen Jahren für die biochemische Analyse bekannt. Lateral Flow Assays (LFA) nutzen den Effekt der Antikörper-Antigen Reaktion aus. Zusätzlich wird die zu analysierende Probe (Lösung) durch Kapillärkräfte über die Sensoroberfläche gezogen. Zum Nachweis von Analyten mittels LFA kann beispielsweise ein direkter, kompetitiver Immunoassay auf einem Nitrozellu- losestreifen ausgeführt werden, wobei die zu analysierende Probe aufgrund von Kapillarkräften durch den gesamten Nitrozellulosestreifen durchgezogen wird. Die Zone, in der der Anti-Analyt-Antikörper immobilisiert wurde, dient als Detektionszone für den Streifentest. Ein Beispiel für einen LFA-Assay zum Nachweis von Mykotoxinen (z.B. Deoxynivalenol) ist der "Reveal-Assay" (Testkassette) der Firma Neogen, Lansing, MI, USA mit dem dazugehörigen Auslesegerät "AccuScan". Die Kartusche wird in das Auslesegerät eingeführt und das Gerät nimmt ein Bild des Ergebnisbereichs des Streifentests. Das Auslesegerät interpretiert das Ergebnisbild und, wenn eine Linie erkannt worden ist, wird eine Bewertung abgegeben. Das Gerät eliminiert die Subjektivität der Interpretation und gibt eine objektive, nachvollziehbare Dokumentation des Testergebnisses. Der beschriebene Test ist einfach und relativ schnell durchzuführen und kommt ohne aufwändige Auslesegeräte aus. Nachteilig ist, dass das Verfahren nur einen qualitativen oder allenfalls semiquantitativen Mykoto- xin-Nachweis erlaubt.The Lateral Flow Assay (LFA) technology has also been known for many years for biochemical analysis. Lateral flow assays (LFA) exploit the effect of the antibody-antigen reaction. In addition, the sample to be analyzed (solution) is drawn over the sensor surface by capillary forces. For the detection of analytes by means of LFA, for example, a direct, competitive immunoassay can be carried out on a nitrocellulose strip, wherein the sample to be analyzed is pulled through the entire nitrocellulose strip due to capillary forces. The zone in which the anti-analyte antibody was immobilized serves as a detection zone for the streak test. An example of an LFA assay for the detection of mycotoxins (e.g., deoxynivalenol) is the "Reveal Assay" (test cassette) from Neogen, Lansing, MI, USA with the associated "AccuScan" reader. The cartridge is inserted into the reader and the device takes a picture of the result range of the strip test. The reader interprets the result image and, if a line has been detected, a rating is given. The device eliminates the subjectivity of the interpretation and gives an objective, traceable documentation of the test result. The test described is simple and relatively fast to carry out and does not require elaborate read-out devices. The disadvantage is that the method only allows qualitative or semiquantitative mycotoxin detection.
WO 2007/079893 beschreibt ein Verfahren zum Schnell-Nachweis von Mykotoxin bei dem eine geträgerte Substanzbibliothek von immobilisierten Bindungspartnem für Mykotoxine und/oder Sonden für Mykotoxine in räumlich getrennten Messbereichen auf der Oberfläche eines Dünnschichtwellenleiters aufgetragen wird, eine Mykotoxin und Sonden dieser Mykotoxin enthaltende Probe in Kontakt zu den immobilisierten Bin- dungspartner gebracht wird und die Reaktion der immobilisierten Bindungspartner mit den Mykotoxinen und/oder Erkennungselementen der Mykotoxine anhand einer Signaländerung im Evaneszenzfeld, das heißt an der Grenzfläche zum Wellenleiter detektiert wird. Von besonderem Vorteil ist bei dem Verfahren, dass auf ein Abwaschen fluoreszent markierter Bindungspartner oder einer markierte Bindungspartner enthaltenden Probe bzw. Lösung vor der Detektion eines Signals eingeschränkt oder sogar völlig verzichtet werden kann. Dies ermöglicht sowohl eine Zeitersparnis bei der Analyse als auch auch eine Vereinfachung der Durchführung, da auch auf die Bereitstellung von Waschlösungen verzichtet werden kann. Die Signalintensität wird anhand einer Bildaufhahme des Assays mittels einer geeigneten Software ermittelt, ebenso wie die Berechnung der in der Probe vorliegenden Menge der Mykotoxine. Es ist allerdings aus dem Stand der Technik bekannt, dass eine geeignete Refenzierungsmethode für die Zuverlässigkeit der quantitativen Analyse vorteilhaft ist. Eine solche Referenzierungsmethode wird in WO 2007/079893 nicht beschrieben.WO 2007/079893 describes a method for rapid detection of mycotoxin in which a supported substance library of immobilized binding partners for mycotoxins and / or probes for mycotoxins in spatially separated measurement areas is applied to the surface of a thin-film waveguide, a mycotoxin and probes containing this mycotoxin sample in Contact with the immobilized binding partners is brought and the reaction of the immobilized binding partner with the mycotoxins and / or recognition elements of mycotoxins is detected by a signal change in the evanescent field, that is at the interface to the waveguide. It is of particular advantage in the method that limited to a washing away fluorescently labeled binding partner or a labeled binding partner containing sample or solution prior to the detection of a signal or even completely can be omitted. This allows both a time savings in the analysis as well as a simplification of the implementation, since it can be dispensed with the provision of washing solutions. The signal intensity is determined by taking an image of the assay using suitable software, as well as calculating the amount of mycotoxins present in the sample. However, it is known from the prior art that a suitable method of refinement is advantageous for the reliability of the quantitative analysis. Such a referencing method is not described in WO 2007/079893.
Im Stand der Technik wird die Nutzung von einem oder mehreren Messbereichen für die Kalibrierung eines Assays beschrieben. In WO 01/13096 z.B. werden Messbereiche zur Referenzierung gleicher chemischer oder - - optischen Parameter (beispielweise die Intensität des lokal verfügbaren Anregungslichts) in mehreren über die Sensorplattform verteilten Probenbehältnissen verwendet, so dass die örtliche Verteilung besagter Parameter auf der Sensorplattform bestimmt werden kann. Die Anzahl und Position der Messbereiche zur Referenzie- rung in der o. g. Anordnung von Messbereichen ist beliebig.The prior art describes the use of one or more ranges for the calibration of an assay. In WO 01/13096, for example, measuring ranges for referencing the same chemical or Optical parameters (for example the intensity of the locally available excitation light) are used in a plurality of sample containers distributed over the sensor platform, so that the local distribution of said parameters on the sensor platform can be determined. The number and position of the measuring ranges for referencing in the above-mentioned arrangement of measuring ranges is arbitrary.
EP-A 0093 613 beschreibt ein Verfahren zur Kalibrierung eines Assays zur Quantifizierung eines Targetmoleküls in einer Probeflüssigkeit mittels eines Sensors basierend auf Fluoreszenzanregung im evaneszenten Feld eines optischen Wellenleiters, der einen ersten Messbereich (Messungsbereich) zur spezifischen Bindung eines ersten Labels aufweist, wobei dieses erste Label in einer Menge, die von der Anwesenheit eines Analy- ten in der Probe abhängt, benutzt wird, und einen zweiten Messbereich (Kalibrierungsbereich) zur Bindung eines zweiten Labels, wobei die Bindung des zweiten Labels von der Anwesenheit des Analyten in der Probe nicht beeinflusst wird. Dabei werden in den Messungsbereichen und Kalibrierungsbereichen verschiedene Bindungspaare verwendet, die aber ähnlicher Natur sind. Die Quantität des zweiten Labels im Kalibrierungsbereich während des Assays gibt einen Signalwert für eine vordefinierte Konzentration des Analyten innerhalb eines Konzentrationsbereichs. Beide Messbereiche sind dicht aneinander platziert, auf der gleichen Grundstruktur, um von möglichen lokalen Variationen des Sensors verursachte Differenzen zu minimieren. Der Signalwert des Messungsbereiches wird durch den Signalwert des dicht aneinander platzierten Kalibrierungsbereiches dividiert, um die nicht-spezifischen Effekten des Sensors auf das Signal zu korrigieren. Der Aufbau des Sensors und die Richtung des Anregungsstrahls werden nicht näher definiert.EP-A 0093 613 describes a method for calibrating an assay for quantifying a target molecule in a sample liquid by means of a sensor based on fluorescence excitation in the evanescent field of an optical waveguide having a first measurement range (measurement range) for the specific binding of a first label Label in an amount dependent on the presence of an analyte in the sample, and a second measurement range (calibration range) for binding a second label, wherein the binding of the second label does not affect the presence of the analyte in the sample becomes. Different binding pairs are used in the measurement ranges and calibration ranges, but they are of similar nature. The quantity of the second label in the calibration area during the assay gives a signal value for a predefined concentration of the analyte within a concentration range. Both measuring ranges are placed close to each other, on the same basic structure, in order to minimize differences caused by possible local variations of the sensor. The signal value of the measurement range is divided by the signal value of the calibration range placed close to each other to correct the non-specific effects of the sensor on the signal. The structure of the sensor and the direction of the excitation beam are not defined.
WO 2004/023142 beschreibt ein Verfahren zur Kalibrierung eines Assays zur Quantifizierung eines Target- moleküls in einer Probeflüssigkeit mittels eines Sensors basierend auf Fluoreszenzanregung im evaneszenten Feld eines optischen Wellenleiters, auf den Erkennungselemente und Referenzmoleküle (Cy5-BSA, BSA = bovine serum albumine) in getrennten parallelen alternierenden Mikroarrays orthogonal zur Ausbreitungsrichtung des in der Evaneszenzfeld-Sensorplattform geführten Anregungslichts in Messungsspots bzw. Referenzspots gespottet sind. Zur Referenzierung der Signalintensität eines jeden Messungspots wird die Netto- Signalintensität des Messungsspots durch den Mittelwert der Netto-Signalintensitäten der benachbarten Referenzspots derselben Reihe angeordnet in der Ausbreitungsrichtung des Anregungslichts dividiert. Durch diese Referenzierung werden die lokalen Unterschiede der verfügbaren Anregungslichtintensität orthogonal zur Lichtausbreitungsrichtung sowohl innerhalb eines jeden Mikroarray als auch zwischen verschiedenen Mikroarrays kompensiert.WO 2004/023142 describes a method for calibrating an assay for quantifying a target molecule in a sample liquid by means of a sensor based on fluorescence excitation in the evanescent field of an optical waveguide, on the recognition elements and reference molecules (Cy5-BSA, bovine serum albumin) in separated parallel alternating microarrays orthogonal to the propagation direction of guided in the Evaneszenzfeld sensor platform excitation light in Messspots or reference spots are spotted. For referencing the signal intensity of each measurement spot, the net signal intensity of the measurement spot is divided by the average of the net signal intensities of the adjacent reference spots of the same row arranged in the propagation direction of the excitation light. This referencing compensates for the local differences in available excitation light intensity orthogonal to the light propagation direction both within each microarray and between different microarrays.
Bei der Verwendung der im Stand der Technik beschriebenen Methoden der Referenzierung zeigte sich, dass diese nicht zur Referenzierung der Assays im fluidischen System geeignet waren. Es zeigte sich, dass bei der Verwendung gespotteter, fluoreszierender Proteine als Referenz nur diejenigen Schwankungen des Systems ausgeglichen werden können, die auf der Ebene des Sensors stattfinden, wie beispielsweise die Dämpfung des Fluoreszenzlichtes oder Schwankungen im Spotting der Arrays.When using the referencing methods described in the prior art, it was found that they were not suitable for the referencing of the assays in the fluidic system. It has been found that when spotted fluorescent proteins are used as a reference, only those fluctuations of the system which take place at the level of the sensor, such as the attenuation of the fluorescent light or fluctuations in the spotting of the arrays, can be compensated.
Aus dem Stand der Technik bestand die Aufgabe ein kostengünstiges, lagerfähiges und einfach zu bedienendes Mittel zur quantitativen Analyse von Analyten, insbesondere Mykotoxinen, mittels einer auf einem Dünnschichtwellenleiter (PWG-Biochip, PWG = planar wave guide) geträgerten Substanzbibliothek von immobili- sierten Bindungspartnern in räumlich getrennten Messbereichen (Immunoassay) bereitzustellen. Es ist eine weitere Aufgabe der vorliegenden Erfindung, eine absolute Bestimmung, d. h. eine Referenzierung des erzeugten Signals, zu ermöglichen.The object of the prior art was a cost-effective, storable and easy-to-use means for the quantitative analysis of analytes, in particular mycotoxins, by means of a substance library of immobilized on a thin-film waveguide (PWG biochip, PWG = planar wave guide). provided binding partners in spatially separated measuring areas (immunoassay). It is a further object of the present invention to enable an absolute determination, ie a referencing of the generated signal.
Diese Aufgabe wird erfindungsgemäß durch eine mikrofluidische Kartusche zur qualitativen und/oder quanti- tativen Analyse von Analyten, insbesondere von Mykotoxinen, gelöst, die alle für die Durchführung des Testverfahrens benötigten Reagenzien in trockener Form beinhaltet. Die erfindungsgemäße Kartusche weist einen strukturierten Körper auf, in den Kavitäten eingebracht wurden, die miteinander durch Kanäle verbunden sind. Erfindungsgemäß weist die Kartusche mindestens einen Einlass zur Einführung einer Mykotoxine enthaltenden Probeflüssigkeit, mindestens eine Reagenzienkammer, und mindestens eine Detektionskammer auf. In der Reagenzienkammer sind in trockener Form ein oder mehrere markierte Mykotoxinsonden zur Reaktion mit den Mykotoxinen aus der Probeflüssigkeit und markierte Referenzierungsonden zur Reaktion mit einem Referenzierungsantigen untergebracht. Der Boden der Detektionskammer besteht aus einem Dünnschichtwellenleiter (PWG-Biochip), umfassend eine erste optisch transparente Schicht (a) auf einer zweiten optisch transparenten Schicht (b), die einen niedrigerem Brechungsindex als Schicht (a) aufweist, und in der ein optisches Gitter eingebracht ist, wobei das Gitter senkrecht zum Pfad eines Anregungslicht orientiert ist, das mittels des optische Gitter in den Dünnschichtwellenleiter eingekoppelt wird. Auf der Oberfläche des Dünnschichtwellenleiters werden Detektionsreagenzien immobilisiert; und zwar werden in Reihen von räumlich getrennten Messbereichen ein Mykotoxinassay (Immunoassay) in Form einer Substanzbibliothek von immobilisierten Bindungspartnern für Mykotoxine und/oder für Mykotoxinsonden und ein unabhängiger Kontroll-Assay beinhaltend ein immobilisiertes Referenzierungsantigen aufgetragen. Die Arrays werden so auf den PWG-Biochip aufgebracht, dass die Messbereiche parallel zum optischen Gitter in Reihen orientiert sind. In der Richtung des Anregungslichtes oberhalb und unterhalb jeder Reihe von Immunoassay befindet sich eine Reihe des Kontroll- Assays (siehe Fig. 1), so dass eine referenzierte Fluoreszenzintensität des Myko- toxinassay-Messbereiches durch Division der Fluoreszenzintensität des Mykotoxinassay-Messbereiches durch den Mittelwert der Fluoreszenzintensitäten der in der Richtung des Anregungslichtes benachbarten Kontroll- Assay -Messbereiche erhalten werden kann.This object is achieved according to the invention by a microfluidic cartridge for the qualitative and / or quantitative analysis of analytes, in particular of mycotoxins, which contains all the reagents required for carrying out the test method in dry form. The cartridge according to the invention has a structured body into which cavities have been introduced, which are connected to one another by channels. According to the invention, the cartridge has at least one inlet for introducing a sample liquid containing mycotoxins, at least one reagent chamber, and at least one detection chamber. One or more labeled mycotoxin probes for reaction with the mycotoxins from the sample fluid and labeled homing probes for reaction with a referencing antigen are housed in the reagent chamber in dry form. The bottom of the detection chamber consists of a thin-film waveguide (PWG biochip) comprising a first optically transparent layer (a) on a second optically transparent layer (b) having a lower refractive index than layer (a) and in which an optical grating is introduced, wherein the grating is oriented perpendicular to the path of an excitation light, which is coupled by means of the optical grating in the thin-film waveguide. On the surface of the thin-film waveguide, detection reagents are immobilized; Namely, in series of spatially separated measurement areas, a mycotoxin assay (immunoassay) in the form of a substance library of immobilized binding partners for mycotoxins and / or mycotoxin probes and an independent control assay comprising an immobilized homing antigen are applied. The arrays are applied to the PWG biochip in such a way that the measurement areas are aligned in rows parallel to the optical grid. In the direction of the excitation light above and below each series of immunoassay is a series of the control assay (see FIG. 1) such that a referenced fluorescence intensity of the mycotoxin assay range is determined by dividing the fluorescence intensity of the mycotoxin assay range by the mean of the mycotoxin assay range Fluorescence intensities of the adjacent in the direction of the excitation light control assay measuring ranges can be obtained.
Überraschenderweise zeigte sich, dass die Referenzierung der Immunoassays im fluidischen System erheblich verbessert wird, wenn statt des bekannten statischen das erfindungsgemäße dynamische Referenzierungskon- zept verwendet wird. Vorteile der dynamischen Referenzierung sind, dass sowohl Schwankungen im fluidi- sehen System (z.B. Adsorption in den Kanälen, Volumenschwankungen, Variationen der Mengen an Antikörpern im Päd) als auch Schwankungen auf der PWG-Biochip-Oberfläche (z.B. Dämpfung, Variationen im Spotting) ausgeglichen werden können.It has surprisingly been found that the referencing of the immunoassays in the fluidic system is considerably improved if, instead of the known static, the dynamic referencing concept according to the invention is used. Advantages of dynamic referencing are that fluctuations in the fluidic system (eg adsorption in the channels, volume fluctuations, variations in the amounts of antibodies in the pedestal) as well as fluctuations on the PWG biochip surface (eg attenuation, variations in spotting) are compensated can be.
Erster Gegenstand der vorliegenden Erfindung ist daher eine Kartusche zum Nachweis und zur quantitativen Analyse von Analyten in einer Probeflüssigkeit, beinhaltend einen strukturierten Körper, in den Kavitäten eingebracht sind, die miteinander durch Kanäle verbunden sind, wobei die Kartusche mindestens einen Einlass zur Einführung der Analyten enthaltenden Probeflüssigkeit, mindestens eine Reagenzienkammer und mindestens eine Detektionskammer aufweist, wobei - - a. in der Reagenzienkammer eine oder mehrere markierte Analytensonden zur Reaktion mit den Analyten aus der Probeflüssigkeit und eine oder mehreremarkierte Referenzierungsson- den zur Reaktion mit einem Referenzierungsantigen in trockener Form untergebracht sind,The first object of the present invention is therefore a cartridge for the detection and quantitative analysis of analytes in a sample liquid, comprising a structured body, are introduced into the cavities, which are connected to each other through channels, said cartridge containing at least one inlet for introducing the analytes Probeflüssigkeit, at least one reagent chamber and at least one detection chamber, wherein - - a. in the reagent chamber one or more labeled analyte probes for reaction with the analytes from the sample liquid and one or more labeled homing probes for reaction with a referencing antigen in dry form are accommodated,
b. der Boden der Detektionskammer ein Dünnschichtwellenleiter ist, umfassend eine erste op- tisch transparente Schicht (a) auf einer zweiten optisch transparenten Schicht (b), die einen niedrigerem Brechungsindex als Schicht (a) aufweist, wobei in der Schicht (a) oder (b) ein optisches Gitter eingebracht ist, das senkrecht zum Pfad eines Anregungslichtes orientiert ist, das mittels des optischen Gitters in den Dünnschichtwellenleiter eingekoppelt wird,b. the bottom of the detection chamber is a thin-film waveguide comprising a first optically transparent layer (a) on a second optically transparent layer (b) having a lower refractive index than layer (a), wherein in layer (a) or (b ) an optical grating is introduced, which is oriented perpendicular to the path of an excitation light, which is coupled by means of the optical grating in the thin-film waveguide,
c. auf der Oberfläche des Dünnschichtwellenleiters ein Immunoassay in Form einer Substanz- bibliothek von immobilisierten Bindungspartnern für Analyten und/oder für Analytensonden in Reihen von räumlich getrennten Messbereichen und ein unabhängiger Kontroll-Assay beinhaltend das Referenzierungsantigen immobilisiert in Reihen von räumlich getrennten Messbereichen aufgetragen sind undc. on the surface of the thin-film waveguide, an immunoassay in the form of a substance library of immobilized binding partners for analytes and / or for analyte probes in rows of spatially separated measurement areas and an independent control assay comprising the referencing antigen immobilized in rows of spatially separated measurement areas are applied and
d. die jeweiligen Reihe parallel zum optischen Gitter orientiert sind und sich in der Richtung des Anregungslichtes oberhalb und unterhalb jeder Reihe des Immunoassays eine Reihe vond. the respective rows are oriented parallel to the optical grating and in the direction of the excitation light above and below each row of the immunoassay a series of
Kontroll-Assays befindet.Control Assays is located.
Vorzugsweise wird der Kontroll-Assay so ausgewählt, dass das Referenzierungsantigen ein ähnliches Molekulargewicht wie der Analyt hat, und die Referenzierungssonde ähnliche Bindungseigenschaften wie die Analytensonden aufweist (Affinität, Bindungskinetik). Der Kontroll-Assay darf zudem keine Kreuzreaktivität zu den Immunoassays zeigen und das Antigen darf in der untersuchten Matrix natürlicherweise nicht vorkommen.Preferably, the control assay is selected such that the referencing antigen has a molecular weight similar to the analyte, and the homing probe has similar binding properties as the analyte probes (affinity, binding kinetics). The control assay must also show no cross-reactivity to the immunoassays and the antigen must not naturally occur in the matrix examined.
Weiterhin vorteilhaft ist, dass sich das Degradationsverhalten des Kontroll-Assays sowie des Immunoassays ähneln, sodass die Langzeitstabilität der Kalibrationskurve eines Produktionsbatches gegeben ist.It is also advantageous that the degradation behavior of the control assay and the immunoassay are similar, so that the long-term stability of the calibration curve of a production batch is given.
In einer besonderen Ausführungsform der Erfindung sind die Analyten Mykotoxine.In a particular embodiment of the invention, the analytes are mycotoxins.
Bevorzugt wird ein Immunonassay wie in WO 2007/079893 beschrieben verwendet, dessen Inhalt per Referenz integriert wird.Preferably, an immunoassay as described in WO 2007/079893 is used, the content of which is integrated by reference.
Bevorzugt werden als Immunoassay Reihen von Mykotoxin-Protein-Konjugaten, z.B. Mykotoxin-BSA- Konjugate.Preferred immunoassay sequences of mycotoxin-protein conjugates, e.g. Mycotoxin-BSA conjugates.
Beispiele für Kontroll-Assays sind Assays gegen Mykotoxine, die in der untersuchten Matrix natürlicherweise nicht vorkommen. Der Kontrollassay wird vorzugsweise so ausgewählt, dass ein Molekül < 1000 g/mol nachgewiesen wird. Besonders bevorzugt werden auf dem PWG-Biochip ein Kontrollassay für Fluorescein und eine Reihe von Kontroll-Protein-Konjugaten, z.B. Fluorescein-BSA, aufgebracht. - -Examples of control assays are assays against mycotoxins that do not naturally occur in the matrix examined. The control assay is preferably selected to detect a molecule <1000 g / mol. Particularly preferably, a control assay for fluorescein and a number of control protein conjugates, eg fluorescein-BSA, are applied to the PWG biochip. - -
Der PWG-Biochip besteht beispielsweise aus einem Glasträger, der mit einer Schicht aus Tantalpentoxid beschichtet ist. Die Schichtdicke beträgt 40 bis 160 nm, bevorzugt 80 bis 160 nm, besonders bevorzugt 120 bis 160 nm, ganz besonders bevorzugt 155 nm. Der Glasträger enthält ein optisches Gitter mit einer Gittertiefe von 3 bis 60 nm, bevorzugt 5 bis 30 nm, besonders bevorzugt 10 bis 25 nm, ganz besonders bevorzugt 18nm und einer Gitterperiode von 200 bis 1000 nm, bevorzugt 220 bis 500 nm, besonders bevorzugt 318 nm. Bevorzugt weist das Gitter eine einzige Periode auf, das heißt es ist monodiffraktiv.The PWG biochip, for example, consists of a glass carrier which is coated with a layer of tantalum pentoxide. The layer thickness is 40 to 160 nm, preferably 80 to 160 nm, particularly preferably 120 to 160 nm, very particularly preferably 155 nm. The glass substrate contains an optical grating with a grating depth of 3 to 60 nm, preferably 5 to 30 nm, particularly preferably 10 to 25 nm, very particularly preferably 18 nm and a grating period of 200 to 1000 nm, preferably 220 to 500 nm, particularly preferably 318 nm. Preferably, the grating has a single period, that is to say it is monodiffractive.
Die Tantalpentoxid-Oberfläche ist üblicherweise mit Dodecylphosphat in Form einer Monoschicht beschichtet. Auf dieser Oberfläche werden Analyten-Protein-Koηjugate, bevorzugt Mykotoxin-BSA Konjugate und Referenzierungsantigen-Protein-Koηjugate, bevorzugt Fluorescein-BSA-Konjugate, immobilisiert. Zur Im- mobilisierung werden üblicherweise die Protein-Konjugate in Konzentrationen von 0,1 bis 5 mg/ml, bevorzugt 0,2bis 2mg/ml, besonders bevorzugt 0,5 bis 1,5 mg/ml, ganz besonders bevorzugt 1 mg/ml auf die Oberfläche aufgebracht und dort adsorbiert.The tantalum pentoxide surface is usually coated with dodecyl phosphate in the form of a monolayer. Analyte-protein conjugates, preferably mycotoxin-BSA conjugates and homing antigen-protein conjugates, preferably fluorescein-BSA conjugates, are immobilized on this surface. For immobilization, the protein conjugates are usually used in concentrations of 0.1 to 5 mg / ml, preferably 0.2 to 2 mg / ml, more preferably 0.5 to 1.5 mg / ml, most preferably 1 mg / ml applied to the surface and adsorbed there.
Zur Aufbringung der Protein-Konjugate können ein oder mehrere Verfahren verwendet werden, die aus der folgenden Gruppe ausgewählt sind: Ink jet Spotting, mechanischem Spotting mittels Stift oder Feder, micro contact printing, fluidische Kontaktierung der Messbereiche mit den biologischen oder biochemischen oder synthetischen Erkennungselementen durch deren Zufuhr in parallelen oder gekreuzten Mikrokanälen, unter Einwirkung von Druckunterschieden oder elektrischen oder elektromagnetischen Potentialen.For application of the protein conjugates, one or more methods selected from the group consisting of ink jet spotting, mechanical spotting by pen or pen, microcontact printing, fluidic contacting of the measurement regions with the biological or biochemical or synthetic recognition elements may be used their supply in parallel or crossed microchannels, under the effect of pressure differences or electrical or electromagnetic potentials.
Nach Immobilisierung der Protein-Konjugate werden die noch freien Bereiche der PWG-Chipoberfläche durch Behandlung mit BSA passiviert um unspezifische Bindungen zu unterdrücken.After immobilization of the protein conjugates, the still free areas of the PWG chip surface are passivated by treatment with BSA in order to suppress non-specific binding.
Der PWG-Biochip stellt den Boden der Detektionskammer der erfindungsgemäßen Kartusche dar und wird in die Kartusche integriert.The PWG biochip represents the bottom of the detection chamber of the cartridge according to the invention and is integrated into the cartridge.
Die Kartusche besteht aus einem strukturierten Körper, in den Kammern und Kanäle eingebracht werden, wobei die Kammern vorzugsweise im Körper so eingebracht werden, dass sie mindestens auf einer Seite durch das Aufbringen einer Verschlusseinheit gebildet werden. Der strukturierte Körper wird oben und unten mittels einer Verschlusseinheit mit Ausnahme des Einlasses, des Bodens der Detektionskammer und optionalen Lüftungsöffhungen verschlossen. Vorzugsweise wird der Biochip vor der Verschlusseinheit in Position gebracht und wird durch die Verschlusseinheit in Position gehalten. Bevorzugt ist die Verschlusseinheit eine Verschlussfolie.The cartridge consists of a structured body, are introduced into the chambers and channels, the chambers are preferably introduced into the body so that they are formed on at least one side by the application of a closure unit. The structured body is closed at the top and bottom by means of a closure unit with the exception of the inlet, the bottom of the detection chamber and optional ventilation openings. Preferably, the biochip is positioned in front of the closure unit and held in position by the closure unit. The closure unit is preferably a closure film.
Vorzugsweise wird in den Kanälen und in den Kammern ein präzise definiertes Volumen an Probeflüssigkeit befördert, was durch die Gestaltung der Kanäle und der Kammer und den Einsatz eines geeigneten Mittels zur Beförderung der Probeflüssigkeit ermöglicht wird. Dabei können Reaktionszeiten ebenfalls genau kontrolliert werden, was zur besseren Reproduzierbarkeit der Analyse beiträgt. Durch das passende Design der Kammer und der Kanäle wird ein optimales Flussprofil mit reduziertem Totvolumen und ggf. optimalem Kontakt mit den immobilisierten Detektionsreagenzien gewährleistet. Die Kanäle verbinden den Einlass, die Reagenzienkammer und die Detektionskammer miteinander und besitzen üblicherweise einen Durchmesser von 0,1 bis 2,5 mm, bevorzugt 0,5 bis 1,5 mm, besonders bevorzugt 1 mm.Preferably, a precisely defined volume of sample liquid is conveyed in the channels and in the chambers, which is made possible by the design of the channels and the chamber and the use of a suitable means for transporting the sample liquid. Reaction times can also be precisely controlled, which contributes to better reproducibility of the analysis. The proper design of the chamber and channels ensures an optimal flow profile with reduced dead volume and possibly optimal contact with the immobilized detection reagents. The channels connect the inlet, the reagent chamber and the detection chamber with each other and usually have a diameter of 0.1 to 2.5 mm, preferably 0.5 to 1.5 mm, particularly preferably 1 mm.
In einer besonderen Ausführungsform der Kartusche weist die Reagenzienkammer ein Reagenzien-Pad auf, auf dem die Analyten- und Referenzierungsonden insb. Antikörper für Mykotoxine und Fluorescein untergebracht sind.In a particular embodiment of the cartridge, the reagent chamber has a reagent pad, on which the analyte and Referenzierungsonden esp. Antibodies for mycotoxins and fluorescein are housed.
Das Reagenzien-Pad wird so ausgewählt, dass es die Anforderungen der Detektionskammer bezüglich des geforderten Flüssigkeitsvolumens der überstehenden Lösung und der Konzentration der Einzelkomponenten in dieser Lösung erfüllt.The reagent pad is selected to meet the requirements of the detection chamber with respect to the required liquid volume of the supernatant solution and the concentration of the individual components in this solution.
Das Reagenzien-Pad besteht üblicherweise aus einem faserartigen oder porösen Material z.B. feinen Partikeln oder Gewebe, in das Reagenzien (adsorbiert auf, fixiert auf, dispergiert in, eingetrocknet in) untergebracht wurden. Ein bevorzugtes Reagenzien-Pad besteht aus Glas oder Polymeren wie z.B. Zellulose. Z.B. werden Reagenzien-Pads verwendet, die auch in sog. Lateral Flow Assays verwendet werden und in verschiedenen Formen kommerziell erhältlich sind.The reagent pad is usually made of a fibrous or porous material, e.g. fine particles or tissue into which reagents (adsorbed on, fixed on, dispersed in, dried in) were placed. A preferred reagent pad is made of glass or polymers, e.g. Cellulose. For example, Reagent pads are used, which are also used in so-called lateral flow assays and commercially available in various forms.
Eine bevorzugte Reagenzienkammer benötigt ein Flüssigkeitsvolumen von 10 bis 100 μl, bevorzugt 20 bis 60 μl, besonders bevorzugt 40 μl und darin gelöste Analyten- und Referenzierungsonden in einer Konzentration von 10"7 M bis 10"10 M, bevorzugt nanomolare Konzentrationen.A preferred reagent chamber requires a liquid volume of 10 to 100 .mu.l, preferably 20 to 60 .mu.l, more preferably 40 .mu.l and analyte and Referenzierungsonden dissolved therein in a concentration of 10 "7 M to 10 " 10 M, preferably nanomolar concentrations.
Zum Befüllen dieser Reagenzienkammer wird das Reagenzien-Pad ausgewählt, das bevorzugt aus extra thick glass filters der Firma PaIl Corporation (Porengröße 1 μm, typische Dicke 1270 μm (50 mils), typisches Was- ser Flussrate 210 mL/min/cm2 bei 30 kPa) besteht, wobei zwei kreisrunde Filterstücke mit einem passenden Durchmesser (üblicherweise von 5 bis 10 mm) übereinander gestapelt werden. Das resultierende Reagenzien- Pad wird üblicherweise mit ca. 100 μl der Lösung imprägniert, die die fluoreszenzmarkierten Sonden enthält, sowie üblicherweise weitere Komponenten zur Unterstützung der Imprägnierung. Die Imprägnierung erfolgt z.B. durch Eintrocknen oder Lyophilisieren.To fill this reagent chamber, the reagent pad is selected, preferably from extra thick glass filters from Paill Corporation (pore size 1 μm, typical thickness 1270 μm (50 mils), typical water flow rate 210 mL / min / cm 2 at 30 kPa), wherein two circular filter pieces with a suitable diameter (usually from 5 to 10 mm) are stacked on top of each other. The resulting reagent pad is usually impregnated with about 100 μl of the solution containing the fluorescently labeled probes and usually other components to aid in impregnation. The impregnation is carried out, for example, by drying or lyophilization.
Das Reagenzien-Pad wird üblicherweise in der Kartusche so betrieben, dass es mit ca. 80 μl Probeflüssigkeit (z.B. Mykotoxin-Extrakt) benetzt wird.The reagent pad is usually operated in the cartridge so that it is wetted with about 80 μl of sample fluid (e.g., mycotoxin extract).
Nach einer Präinkubationszeit von 1 bis 10 min werden üblicherweise 20 bis 60 μl der Lösung in die Detektionskammer befördert.After a preincubation time of 1 to 10 minutes usually 20 to 60 .mu.l of the solution are conveyed into the detection chamber.
Eine genaue Kontrolle der Volumen ist bei der vorliegenden Erfindung vorteilhaft aber nicht erforderlich, da Variationen zwischen den verschiedenen Kartuschen durch das erfindungsgemäße Referenzierungs verfahren ausgeglichen werden können.Accurate volume control is advantageous but not required in the present invention because variations between the various cartridges can be accommodated by the inventive referencing method.
Weiterer Gegenstand der vorliegenden Erfindung ist ein Verfahren zum Nachweis von Analyten, insbesondere Mykotoxinen, mittels der erfindungsgemäßen Kartusche. Zweiter Gegenstand der vorliegenden Erfindung ist ein Verfahren zur quantitativen Analyse von Analyten, das folgende Schritte umfasst:Another object of the present invention is a method for the detection of analytes, in particular mycotoxins, by means of the cartridge according to the invention. Second object of the present invention is a method for the quantitative analysis of analytes, comprising the following steps:
a. optionale Extraktion der Analyten aus einer Matrix in eine Probeflüssigkeit,a. optional extraction of the analytes from a matrix into a sample liquid,
b. Durchführung des Assays in der Kartusche nach einem der Ansprüche 1 bis 7, wobei nach Einfuhrung der Probeflüssigkeit in die Kartusche, die Probeflüssigkeit in die Reagenzienkammer befördert wird und sich mit dort aufgebrachten markierten Sonden vermischt bzw. reagiert, dannb. Carrying out the assay in the cartridge according to one of claims 1 to 7, wherein after introduction of the sample liquid into the cartridge, the sample liquid is conveyed into the reagent chamber and mixed or reacted with labeled probes applied there, then
c. Beförderung der Probeflüssigkeit in die Detektionskammer und Reaktion der Analyten und/oder die markierten Sonde mit dem Immunoassay und Kontroll-Assay, gefolgt vonc. Transport of the sample liquid into the detection chamber and reaction of the analytes and / or the labeled probe with the immunoassay and control assay, followed by
d. Ausleuchtung des Dünnschichtwellenleiters zur Anregung der markierten Sonden des Im- munoassays und Kontroll-Assays zur Fluoreszenz und Aufnahme eines Fluoreszenzbildes, dannd. Illumination of the thin-film waveguide for excitation of the labeled probes of the immunoassay and control assay for fluorescence and recording of a fluorescence image, then
e. Berechnung der referenzierten Fluoreszenzintensitäten des Immunoassays anhand des Kontroll-Assays, wobei die referenzierte Fluoreszenzintensität eines jeden Immunoassay- Messbereiches durch Division der Fluoreszenzintensität des Immunoassay-Messbereiches durch den Mittelwert der Fluoreszenzintensitäten der in der Richtung des Anregungslichtes benachbarten Kontroll-Assay-Messbereiche berechnet wird, unde. Calculating the referenced fluorescence intensities of the immunoassay using the control assay, wherein the referenced fluorescence intensity of each immunoassay measurement region is calculated by dividing the fluorescence intensity of the immunoassay measurement region by the mean of the fluorescence intensities of the control assay measurement regions adjacent in the direction of the excitation light;
f. Berechnung und Anzeige der Analyten- Werte durch Bezug auf eine Kalibrationskurve.f. Calculation and display of the analyte values by reference to a calibration curve.
Befinden sich die Mykotoxine in einer festen Matrix, so wird diese in einem optionalen ersten Schritt des er- findungsgemäßen Verfahrens in der Regel zerkleinert, anschließend werden die Mykotoxine mit einem geeigneten Lösungsmittel aus der Matrix extrahiert. Beispiele für Extraktionsmittel sind wässrige Lösungen von Methanol, Ethanol oder Acetonitril. Beispiele für feste Matrices sind Weizen, Mais, Gerste, Roggen, Erdnüsse, Haselnüsse etc. Enthält der Extrakt mehr als 10% des nichtwässrigen Lösungsmittels, so ist in der Regel ein Verdünnungsschritt vor dem Befüllen der Kartusche erforderlich. Flüssige Matrices (Milch, Obstsaft, Wein etc.) können direkt oder nach einer geeigneten Verdünnung in die Kartusche eingefüllt werden.If the mycotoxins are present in a solid matrix, they are usually comminuted in an optional first step of the process according to the invention, after which the mycotoxins are extracted from the matrix with a suitable solvent. Examples of extractants are aqueous solutions of methanol, ethanol or acetonitrile. Examples of solid matrices are wheat, corn, barley, rye, peanuts, hazelnuts, etc. If the extract contains more than 10% of the nonaqueous solvent, a dilution step is usually required before filling the cartridge. Liquid matrices (milk, fruit juice, wine etc.) can be filled into the cartridge directly or after a suitable dilution.
Li einem weiteren Schritt füllt der Nutzer den Extrakt oder die Probenlösung in die Kartusche ein und verschließt die Kartusche. Die Kartusche wird anschließend in ein Auslesegerät eingeführt. Das Auslesegerät enthält eine Pumpe, die Luft in die Kartusche pumpt und so die Lösung von dem Probeneinlass in die Reaktionskammer befördert, wo sie das dort aufgebrachte Reagenzien-Pad benetzt.In a further step, the user fills the extract or the sample solution in the cartridge and closes the cartridge. The cartridge is then inserted into a reader. The reader includes a pump that pumps air into the cartridge, transporting the solution from the sample inlet into the reaction chamber where it wets the reagent pad applied there.
Bei der Benetzung des Reagenzien-Pads werden die Antikörper mit Hilfe des Extraktes aus dem Reagenzien- Pad herausgelöst und so mit dem Extrakt gemischt.When wetting the reagent pad, the antibodies are removed from the reagent pad using the extract and mixed with the extract.
Die Inkubationszeit des Extraktes im Reagenzien-Pad beträgt bevorzugt 1 bis 20 min, besonders bevorzugt 3 bis 7 min. Die Pumpe pumpt nun abermals Luft in die Kartusche und bewegt dadurch das Flüssigkeitsvolu- - - men in die Detektionskammer über dem PWG-Biochip. Abermals erfolgt ein Inkubationsschritt, der üblicherweise 1 bis 100 min, bevorzugt 5 bis 15 min dauert.The incubation time of the extract in the reagent pad is preferably 1 to 20 minutes, more preferably 3 to 7 minutes. The pump now again pumps air into the cartridge and thereby moves the liquid volume - into the detection chamber above the PWG biochip. Again, there is an incubation step, which usually takes 1 to 100 minutes, preferably 5 to 15 minutes.
Vorzugsweise wird die Kartusche über die Dauer des Verfahrens auf eine Temperatur, die bevorzugt 20 bis 37 0C, besonders bevorzugt 25 0C beträgt, temperiert.Preferably, the cartridge over the duration of the process to a temperature which is preferably 20 to 37 0 C, more preferably 25 0 C, tempered.
Nach Inkubation der markierten Antikörper auf dem PWG-Biochip wird ein Laserstrahl in das optische Gitter eingekoppelt. Durch die flächige Ausleuchtung des PWG-Biochips werden die markierten Antikörper zur Fluoreszenz angeregt. Mit Hilfe einer Kamera und eines geeigneten Fluoreszenzfϊlters wird das Fluoreszenzbild des Biochips aufgenommen.After incubation of the labeled antibodies on the PWG biochip, a laser beam is coupled into the optical grating. The extensive illumination of the PWG biochip stimulates the labeled antibodies to fluoresce. With the help of a camera and a suitable fluorescence filter, the fluorescence image of the biochip is recorded.
Eine bildauswertende Software, die im Computer des Auslesegerätes installiert ist, bestimmt nun die Fluores- zenzintensität der Mykotoxin- und der Kontroll-Assay-Messbereiche. Durch Division der Fluoreszenzintensität des Mykotoxin-Assay-Messbereiches durch den Mittelwert der Fluoreszenzintensitäten der in der Richtung des Anregungslichtes benachbarten Kontroll-Assay-Messbereiche wird eine referenzierte Fluoreszenzintensität des Mykotoxin-Assay-Messbereiches erhalten.An image-evaluating software, which is installed in the computer of the reader, now determines the fluorescence intensity of the mycotoxin and control assay ranges. By dividing the fluorescence intensity of the mycotoxin assay measurement range by the mean of the fluorescence intensities of the control assay measurement regions adjacent in the direction of the excitation light, a referenced fluorescence intensity of the mycotoxin assay measurement region is obtained.
Die quantitative Beziehung zwischen den referenzierten Fluoreszenzintensitäten der Mykotoxin-Assay- Messbereiche und der Konzentration eines Mykotoxins in der Lösung, die in die Kartusche pipettiert wurde, wird üblicherweise durch Aufnahme von Kalibrationskurven ermittelt. Die resultierenden mathematischen Beziehungen werden auf dem Computer des Auslesegerätes gespeichert.The quantitative relationship between the referenced fluorescence intensities of the mycotoxin assay ranges and the concentration of a mycotoxin in the solution pipetted into the cartridge is usually determined by taking calibration curves. The resulting mathematical relationships are stored on the computer of the reader.
Wird eine Probe gemessen, so wird nach Aufnahme des Fluoreszenzbildes die referenzierte Fluoreszenzintensität ermittelt und durch Bezug auf die Kalibrationskurve die entsprechende Mykotoxin-Konzentration er- rechnet. Der Mykotoxin- Wert wird auf dem Bildschirm des Auslesegerätes dargestellt.If a sample is measured, the referenced fluorescence intensity is determined after taking the fluorescence image and the corresponding mycotoxin concentration is calculated by reference to the calibration curve. The mycotoxin value is displayed on the screen of the reader.
Die erfindungsgemäße Vorrichtung und das erfindungsgemäße Verfahren werden anhand der folgenden Beispiele und Zeichnungen näher erläutert, ohne sich darauf zu begrenzen.The device according to the invention and the method according to the invention are explained in more detail with reference to the following examples and drawings without being limited thereto.
Zeichnungen:Drawings:
Fig. 1 : Konstruktion des Mykotoxin-Arrays Fig. 2: Aufbau der KartuscheFig. 1: Construction of the mycotoxin array Fig. 2: Structure of the cartridge
Fig. 3: PWG-Biochip Seitenansicht Fig. 4: Dimensionen des PWG-Biochips.Fig. 3: PWG biochip side view Fig. 4: Dimensions of the PWG biochip.
Bezugszeichen:Reference numerals:
1 Kartusche 2 Einlass1 cartridge 2 inlet
3 Kanal3 channel
4 Reagenzienkammer mit Reagenzien-Pad4 Reagent chamber with reagent pad
5 Detektionskammer 6 PWG-Biochip5 detection chamber 6 PWG biochip
7 Gitter7 grids
8 Glassplatte8 glass plate
9 Wellenleitende Schicht 10 Monoschicht aus Dodecylphosphat / Haftvermittlungsschicht9 Waveguiding layer 10 Monolayer of dodecyl phosphate / adhesion promoting layer
11 Referenzspots / Kontroll-Assay11 reference spots / control assay
12 Mykotoxin-BSA-Konjugate-Spots / Immunoassay12 mycotoxin-BSA conjugate spots / immunoassay
13 Referenzspots / Kontroll-Assay13 reference spots / control assay
14 BSA14 BSA
Die Kartusche (1) besteht aus einem strukturierten Körper, in den Kanäle und Kavitäten eingebracht wurden.The cartridge (1) consists of a structured body, were introduced into the channels and cavities.
Beispielweise wurde die erfindungsgemäße Kartusche in einem Spritzgussverfahren produziert. Der Körper besteht einer Platte aus schwarzem Polyoxymethylen (POM), in der die Kanäle und Kammern aufgebohrt und abgefräst wurden.For example, the cartridge according to the invention was produced in an injection molding process. The body consists of a black polyoxymethylene (POM) plate in which the channels and chambers have been drilled and milled off.
Die Kartusche (1) umfasst einen Einlass (2) zur Eingabe einer Probenfiüssigkeit mit dem zu detektierenden Analyten in eine Probenkammer der Kartusche (1), eine Reagenzienkammer mit einem Reagenzien Päd (4), in die die Probeflüssigkeit über einen Kanal (3) befördert wird, und eine Detektionskammer (5), in die die Probeflüssigkeit über einen weiteren Kanal (3) befördert wird und die einen PWG-Biochip (6) umfasst.The cartridge (1) comprises an inlet (2) for introducing a Probenfiüssigkeit with the analyte to be detected in a sample chamber of the cartridge (1), a reagent chamber with a reagent Päd (4), in which the sample liquid via a channel (3) and a detection chamber (5) into which the sample liquid is conveyed via a further channel (3) and which comprises a PWG biochip (6).
In der Reagenzienkammer (4) befanden sich mit einem Fluoreszenzfarbstoff markierten Antikörper, die für Mykotoxine aus der Probeflüssigkeit spezifisch sind, und markierte Antikörper, die spezifisch für die Fluores- cein sind, imprägniert auf dem Reagenzien-Pad.In the reagent chamber (4) were antibodies labeled with a fluorescent dye specific for mycotoxins from the sample liquid and labeled antibodies specific for the fluoresceins impregnated on the reagent pad.
Sowohl der PWG-Biochip (6) als der Reagenzien-Pad wurden zwischen zwei Polyolefinfolien, in der POM- Platte festgehalten, die auch als Verschlussfolien zur Dichtung der Testkassette dienten. Die obere Verschlussfolie war 180 μm dick und die untere Verschlussfolie war 80 μm dick.Both the PWG biochip (6) and the reagent pad were held between two polyolefin sheets, in the POM plate, which also served as sealing sheets to seal the test cassette. The top closure film was 180 μm thick and the bottom closure film was 80 μm thick.
Die untere Folie wies im Bereich des PWG-Biochip (6) ein Fenster auf, das freien Zugang zu der Messregion des PWG-Biochips (6) gewährte.The bottom foil had a window in the area of the PWG biochip (6) which allowed free access to the measurement region of the PWG biochip (6).
Die Probeflüssigkeit wurde bei Testbeginn durch den Einlass (2) in die Probenkammer eingebracht und der Einlass (2) wurde mit einem geeigneten Deckel luftdicht verschlossen. Mit Hilfe der Beförderungseinheit, wurde an dem Einlass ein definiertes Luftvolumen in die Kartusche (1) eingebracht. Dieses Luftvolumen verdrängte die Probenflüssigkeit, so dass sie in die Reagenzienkammer (4) strömte und das Reagenzien-Pad komplett benetzte.The sample liquid was introduced into the sample chamber through the inlet (2) at the start of the test, and the inlet (2) was sealed airtight with a suitable lid. With the aid of the transport unit, a defined volume of air was introduced into the cartridge (1) at the inlet. This volume of air displaced the sample fluid so that it flowed into the reagent chamber (4) and completely wetted the reagent pad.
Durch Einleitung der Probeflüssigkeit in die Reagenzienkammer (4) wurden die Antikörper gelöst, vermischten sich mit der Probeflüssigkeit und gingen mit den in der Probeflüssigkeit enthaltenen Mykotoxinen eine spezifische Bindung ein (Mykotoxin-Antikörper-Konjugat). Dabei wurden die freien Bindungsstellen der Antikörper mit zunehmender Menge an Mykotoxinen in der Probeflüssigkeit zunehmend gesättigt. - -By introducing the sample liquid into the reagent chamber (4), the antibodies were dissolved, mixed with the sample liquid, and bound with the mycotoxins contained in the sample liquid (mycotoxin-antibody conjugate). The free binding sites of the antibodies were increasingly saturated with increasing amount of mycotoxins in the sample liquid. - -
Nach einer gewissen Verweilzeit (10 Minuten) bei einer Temperatur von 25 0C wurde die Probeflüssigkeit enthaltend Mykotoxin-Antiköφer-Konjugate und die Antikörper für das Fluorescein in einem nächsten Schritt in die Detektionskammer (5) befördert.After a certain residence time (10 minutes) at a temperature of 25 0 C, the sample containing liquid was mycotoxin Antiköφer conjugates and conveys the antibody for fluorescein in a next step in the detection chamber (5).
In der Detektionskammer (5) wurde der Verlauf oder der Endpunkt der biochemischen Nachweisreaktion de- tektiert.In the detection chamber (5), the course or the end point of the biochemical detection reaction was detected.
Die Detektionskammer (5) wurde mit der Probeflüssigkeit komplett gefüllt. Das Kanalsystem wurde komplett entlüftet. Die Entlüftung des kompletten Kanalsystems fand über Entlüftungsόffhungen, die in der oberen Verschlussfohe aufgebracht waren, statt.The detection chamber (5) was completely filled with the sample liquid. The duct system was completely vented. The venting of the complete channel system took place via venting holes applied in the upper closure panel.
Die Detektionskammer (5) umfasste einen PWG-Biochip (6). Der PWG-Biochip (6) ist in Fig. 2 schematisch in Aufsicht und in Fig. 3 schematisch in Seitenansicht dargestellt.The detection chamber (5) comprised a PWG biochip (6). The PWG biochip (6) is shown schematically in plan view in FIG. 2 and schematically in side view in FIG.
Der PWG-Biochip (6) in der Detektionskammer (5) bestand aus einer 10 mm x 12 mm Glassplatte (8) mit einer Dicke von 0.7 mm (12.0 +/- 0.05 mm x 10.0 +/- 0.05 mm x 0.70 +/- 0.05 mm). Auf einer Seite des PWG- Chips (6) befanden sich eine 155 nm dünne wellenleitende Schicht (9) aus Ta2O5 (Tantalpentoxid). Die Messregion des Chips bestand aus einem zentralen 10 mm x 6 mm viereckigen Bereich. Parallel zu dieser Messre- gion befindet sich ein 500 um breites sichelförmiges Band: das Gitter (7) zur Einkopplung des Anregungs- hchts. Die Positionsgenauigkeit des Gitters (7) zu den Kanten betrug +/- 0.05 mm. Die Gittertiefe betrug 18 nm und die Gitterpenode 318 nm mit einer Duty Cycle von 0,5.The PWG biochip (6) in the detection chamber (5) consisted of a 10 mm x 12 mm glass plate (8) with a thickness of 0.7 mm (12.0 +/- 0.05 mm x 10.0 +/- 0.05 mm x 0.70 +/- 0.05 mm). On one side of the PWG chip (6) was a 155 nm thin waveguiding layer (9) of Ta 2 O 5 (tantalum pentoxide). The measuring region of the chip consisted of a central 10 mm x 6 mm square area. Parallel to this measurement region is a 500 μm wide crescent-shaped band: the grating (7) for coupling the excitation beam. The positional accuracy of the grating (7) to the edges was +/- 0.05 mm. The grating depth was 18 nm and the grating 318 nm with a duty cycle of 0.5.
Auf den PWG-Biochip (6) wurde eine Monoschicht aus Dodecylphosphat als Haftvermittlungsschicht (10) aufgebracht. Auf der Haftvermittlungsschicht (10) befanden sich Mykotoxin-BSA-Konjugate adsorptiv in Form eines Immunoassays (12) in Form von Reihen von Spots parallel zum optischen Gitter (Arrays) aufge- tropft/immobihsiert. Oberhalb und unterhalb jeder Reihe von Mykotoxin-BSA-Koηjugaten-Spots (Immuno- assay (12)) befanden sich eine Reihe von BSA-Fluorescein-Spots (Kontroll-Assay/Referenzspots (11, 13)) (Fig 1). Die freien Flächen zwischen den Immunoassays (12) und Kontroll-Assays war mit BSA (14) geblockt (Passivierung).On the PWG biochip (6), a monolayer of dodecyl phosphate was applied as a primer layer (10). On the adhesion-promoting layer (10), mycotoxin-BSA conjugates were adsorbed in the form of an immunoassay (12) in the form of rows of spots dripped / immobiZed parallel to the optical grid (arrays). Above and below each series of mycotoxin-BSA conjugate spots (immunoassay (12)) were a series of BSA fluorescein spots (control assay / reference spots (11, 13)) (Figure 1). The free areas between the immunoassays (12) and control assays were blocked with BSA (14) (passivation).
In der Detektionskammer (5) gelangen die Mykotoxm-Antikörper-Konjugat und ggf. Antikörper mit freien Bindungsstellen sowie die Antikörper für das Fluorescein zu den Immunoassay (12) aus immobilisierten Ana- lyten-BSA-Konjugaten bzw. dem Kontroll-Assay (11, 13) auf dem PWG-Biochip (6). Antikörper mit freien Bindungsstellen gingen eine spezifische Bindung mit den entsprechenden immobilisierten Analyten-BSA- Konjugaten ein.In the detection chamber (5), the mycotoxic antibody conjugate and optionally antibodies with free binding sites and the antibodies for the fluorescein pass to the immunoassay (12) from immobilized analyte-BSA conjugates or the control assay (11, 13) on the PWG biochip (6). Antibodies with free binding sites entered into specific binding with the corresponding immobilized analyte-BSA conjugates.
Je mehr Antikörper mit freien Bindungsstellen in der Lösung vorhanden war, das heißt je geπnger der Anteil des korrespondierenden Analyten in der Probeflüssigkeit war, desto mehr mit einem Fluoreszenz-Farbstoff markierte Antikörper wurden auf dem PWG-Biochip gebunden. Die mit Analyten in der Probeflüssigkeit gesättigten Antikörper verblieben in der Lösung. Durch Einkoppelung elektromagnetischer Strahlung in den PWG-Biochip (6) konnten die an die immobilisierten Analyten-BSA-Konjugate gebundenen und mit einem Fluoreszenzfarbstoff markierten Antikörper im evaneszenten Feld des Wellenleiters zur Fluoreszenz angeregt werden. Die in Lösung befindlichen und mit einem Fluoreszenzfarbstoff markierten Antikörper wurden hierbei nicht angeregt. Auf diese Weise wurde eine indirekte Quantifizierung der in der Probeflüssigkeit enthaltenden Mykotoxine erreicht.The more antibody with free binding sites was present in the solution, that is, the more the proportion of the corresponding analyte in the sample liquid was, the more antibodies labeled with a fluorescent dye were bound to the PWG biochip. The antibodies saturated with analytes in the sample liquid remained in the solution. By coupling electromagnetic radiation into the PWG biochip (6), the antibodies bound to the immobilized analyte-BSA conjugates and labeled with a fluorescent dye could be excited to fluoresce in the evanescent field of the waveguide. The antibodies in solution and labeled with a fluorescent dye were not excited in this case. In this way, an indirect quantification of the mycotoxins contained in the sample liquid was achieved.
Durch Division der Fluoreszenzintensität des Mykotoxinspots durch den Mittelwert der Fluoreszenzintensitäten der Referenzspots wurde eine referenzierte Fluoreszenzintensität des Mykotoxinspots erhalten.By dividing the fluorescence intensity of the mycotoxin spot by the mean of the fluorescence intensities of the reference spots, a referenced fluorescence intensity of the mycotoxin spot was obtained.
Die quantitative Beziehung zwischen den referenzierten Fluoreszenzintensitäten der Mykotoxinspots und der Konzentration eines Mykotoxins in der Lösung, die in die Kartusche pipettiert wurde, wurde durch Aufnahme von Kalibrationskurven ermittelt. Die resultierenden mathematischen Beziehungen wurden auf dem Computer des Auslesegerätes gespeichert.The quantitative relationship between the referenced fluorescence intensities of the mycotoxin spots and the concentration of a mycotoxin in the solution pipetted into the cartridge was determined by recording calibration curves. The resulting mathematical relationships were stored on the computer of the reader.
Beispiel 1example 1
Herstellung von Kartuschen zur Bestimmung von Deoxynivalenol CDON) auf einem PWG-BiochipPreparation of cartridges for the determination of deoxynivalenol CDON) on a PWG biochip
24 PWG-Biochips (Unaxis, Liechtenstein) mit den Außenmaßen 10 mm x 12 mm aus Glasversehen mit einer Schicht (155 nm) von Tantalpentoxid, , in das ein optisches Gitter (Gittertiefe 18 ran) eingeschrieben war, wurden gereinigt und mit Dodecylphosphat beschichtet. Mit Hilfe eines Spotters des Typs Nanoplotter (Ge- SIM, Deutschland) wurden Konjugate aus Deoxynivalenol und Bovinem Serum Albumin (DON-BSA, Biopure, Österreich) sowie Konjugate aus Bovinem Serum Albumin und Fluorescein (BSA-FITC, Sigma, Deutschland) auf den Biochip aufgebracht. Die Spots wurden in Form alternierender Reihen von jeweils 16 BSA-FITC-Koηjugat-Spots und BSA-DON-Konjugat-Spots auf den PWG-Biochip aufgebracht, sodass die Reihen jeweils parallel zum optischen Gitter verliefen. Man ließ die Spots eintrocknen und behandelte anschließend mit dem Nebel einer wässrigen BSA-Lösung. Man wusch die PWG-Biochips und ließ sie anschließend trocknen. Die PWG-Biochips wurden mit doppelseitigem Klebeband in Kartuschen eingeklebt. Die Kartuschen enthielten eine Probenkammer für die Probenaufhahme, eine Reagenzienkammer mit einem Glasfaser-Pads und eine Detektionskammer für den PWG-Biochip. Die Kammern waren durch Kanäle miteinander verbunden. Man imprägnierte das Glasfasermaterial mit Lösungen nanomolarer Konzentrationen von Antikörpern, die mit dem Fluoreszenzfarbstoff DY-647 (Dyomics, Deutschland) markiert waren, wobei monoklonale Antikörper gegen Deoxynivalenol und Fluorescein verwendet wurden. Die Antikörper waren in einem Puffer gelöst, der PBS (= phosphate buffered saline), 0,1 % Ovalbumin, 0,05 % Tween und 5 % Sucro- se enthielt. Die erhaltene Reagenzien-Pads wurden im Vakuum getrocknet und anschließend in die Kartuschen eingestempelt. Die Kartuschen wurden beidseitig mit Verschlussfolien verschlossen, um die Kanäle abzudichten. - -Twenty-four PWG biochips (Unaxis, Liechtenstein) with the external dimensions 10 mm x 12 mm glass-lined with a layer (155 nm) of tantalum pentoxide, in which an optical grating (lattice depth 18 ran) was inscribed, were cleaned and coated with dodecyl phosphate. Conjugates of deoxynivalenol and bovine serum albumin (DON-BSA, Biopure, Austria) as well as conjugates of bovine serum albumin and fluorescein (BSA-FITC, Sigma, Germany) were applied to the Nanoplotter (GE SIM, Germany) spotter Biochip applied. The spots were applied to the PWG biochip in alternating rows of 16 BSA-FITC conjugate spots and BSA-DON conjugate spots, respectively, so that the rows ran parallel to the optical grating. The spots were allowed to dry and then treated with the mist of an aqueous BSA solution. The PWG biochips were washed and then allowed to dry. The PWG biochips were glued into cartridges with double-sided adhesive tape. The cartridges contained a sample chamber for sample collection, a reagent chamber with a glass fiber pad and a detection chamber for the PWG biochip. The chambers were connected by channels. The glass fiber material was impregnated with solutions of nanomolar concentrations of antibodies labeled with the fluorescent dye DY-647 (Dyomics, Germany) using monoclonal antibodies against deoxynivalenol and fluorescein. The antibodies were dissolved in a buffer containing PBS (= phosphate buffered saline), 0.1% ovalbumin, 0.05% Tween and 5% sucrose. The resulting reagent pads were dried in vacuo and then stamped in the cartridges. The cartridges were closed on both sides with sealing films to seal the channels. - -
Beispiel 2Example 2
Aufnahme einer Standardkurve (Kalibrationskurve) zur Quantifizierung von DONRecording a standard curve (calibration curve) to quantify DON
Man bereitete Lösungen von DON in Konzentrationen, die von 0 bis 6000 ppb reichten und befüllte 17 separate Kartuschen mit jeweils 200 μl der Lösung. Die Kartuschen wurden verschlossen und anschließend in das Auslesegerät MyToLab (Bayer Technology Services, Deutschland) eingeführt. Das Auslesegerät war so eingestellt, dass die interne Beförderungseinheit des Gerätes die in die Kartusche eingeführte Flüssigkeit zunächst in das Reagenzien-Pad und nach 5 min Präinkubationszeit in die Detektionskammer transportierte. Die Temperatur wurde dabei konstant bei 25 0C gehalten. Nach 10 min Inkubationszeit in der Chipkammer erfolgte die Einkopplung des Lasers in das optische Gitter des PWG-Biochips. Es wurde ein Fluoreszenzbild jedes einzelnen PWG-Biochips bei einer Integrationszeit von 2 bis 3 sec. aufgenommen. Die erhaltenen Fluoreszenzintensitäten für jeden DON-Spot wurden durch den Mittelwert der Fluoreszenzintensitäten des oberhalb und unterhalb des jeweiligen DON-Spots gelegenen BSA-FITC-Spots dividiert. Die Mittelwerte der so referenzierten Fluoreszenzintensitäten aller 16 DON-Spots wurden ermittelt. Die erhaltenen konzentrationsabhängigen, referenzierten Fluoreszenzintensitäten wurden mit Hilfe des Computerprogramms Origin 7G (O- rigin Lab Corporation, USA) durch einen sigmoidalen Fit angepasst.Prepared solutions of DON in concentrations ranging from 0 to 6000 ppb and filled 17 separate cartridges each with 200 ul of the solution. The cartridges were sealed and then introduced into the MyToLab reader (Bayer Technology Services, Germany). The reader was set so that the internal transport unit of the instrument first transported the liquid introduced into the cartridge into the reagent pad and after 5 minutes of preincubation time into the detection chamber. The temperature was kept constant at 25 0 C. After 10 min incubation time in the chip chamber, the laser was coupled into the optical grating of the PWG biochip. A fluorescence image of each individual PWG biochip was recorded at an integration time of 2 to 3 sec. The fluorescence intensities obtained for each DON spot were divided by the average of the fluorescence intensities of the BSA-FITC spot located above and below the respective DON spot. The mean values of the thus referenced fluorescence intensities of all 16 DON spots were determined. The resulting concentration-dependent, referenced fluorescence intensities were adjusted by a sigmoidal fit using the computer program Origin 7G (Origin Lab Corporation, USA).
Beispiel 3Example 3
Messung von DON in künstlich kontaminierten WeizenprobenMeasurement of DON in artificially contaminated wheat samples
Es wurden Weizenkörner gemahlen und das resultierende Mehl mit einer bekannten Menge einer DON- Lösung versehen, die man eintrocknen ließ. Die homogenisierte Probe enthielt 888 mg/kg (ppb) DON. Man extrahierte 5 g der Mehl-Probe mit 25 ml 70 % Methanol, indem man 3 min intensiv schüttelte. Man ließ den Extrakt absetzen und verdünnte den Überstand im Verhältnis 1:3 mit Puffer. Der verdünnte Extrakt wurde in 7 verschiedene Kartusche eingefüllt. Die Kartuschen wurden anschließend wie oben beschrieben in dem Auslesegerät MyToLab vermessen und die referenzierten Fluoreszenzintensitäten der DON-Spots ermittelt. In Relation zu der oben beschriebenen Standardkurve wurden die Konzentrationen von DON in ppb ermittelt, wo- bei Werte von 1042, 757, 710, 660, 431, 728 und 984 ppb erhalten wurden. Der Mittelwert der DON- Bestimmung betrug 760 ppb bei einer prozentualen Standardabweichung von 27 %. Wheat grains were ground and the resulting flour was provided with a known amount of a DON solution, which was allowed to dry. The homogenized sample contained 888 mg / kg (ppb) of DON. 5 g of the flour sample was extracted with 25 ml of 70% methanol by vigorous shaking for 3 min. The extract was allowed to settle and the supernatant was diluted 1: 3 with buffer. The diluted extract was filled in 7 different cartridges. The cartridges were then measured in the read-out device MyToLab as described above and the referenced fluorescence intensities of the DON spots were determined. In relation to the standard curve described above, the concentrations of DON in ppb were determined to obtain values of 1042, 757, 710, 660, 431, 728 and 984 ppb. The mean DON determination was 760 ppb with a percent standard deviation of 27%.

Claims

Patentansprüche claims
1. Kartusche zum Nachweis und zur quantitativen Analyse von Analyten in einer Probeflüssigkeit, beinhaltend einen strukturierten Körper, in den Kavitäten eingebracht sind, die miteinander durch Kanäle verbunden sind, wobei die Kartusche mindestens einen Einlass zur Einführung der Analyten enthal- tenden Probeflüssigkeit, mindestens eine Reagenzienkammer und mindestens eine Detektionskam- mer aufweist, wobeiA cartridge for the detection and quantitative analysis of analytes in a sample liquid, comprising a structured body, are introduced into the cavities, which are connected to each other through channels, wherein the cartridge at least one inlet for introducing the analyte containing sample liquid, at least one Reagent chamber and at least one detection chamber, wherein
a. in der Reagenzienkammer eine oder mehrere markierte Analytensonden zur Reaktion mit den Analyten aus der Probeflüssigkeit und eine oder mehrere markierte Referenzierungsson- den zur Reaktion mit einem Referenzierungsantigen in trockener Form untergebracht sind,a. one or more labeled analyte probes for reaction with the analytes from the sample liquid and one or more labeled homing probes for reaction with a referencing antigen in dry form are accommodated in the reagent chamber,
b. der Boden der Detektionskammer ein Dünnschichtwellenleiter ist, umfassend eine erste optisch transparente Schicht (a) auf einer zweiten optisch transparenten Schicht (b), die einen niedrigerem Brechungsindex als Schicht (a) aufweist, wobei in der Schicht (a) oder (b) ein optisches Gitter eingebracht ist, das senkrecht zum Pfad eines Anregungslichtes orientiert ist, das mittels des optischen Gitters in den Dünnschichtwellenleiter eingekoppelt wird,b. the bottom of the detection chamber is a thin film waveguide comprising a first optically transparent layer (a) on a second optically transparent layer (b) having a lower refractive index than layer (a), wherein in layer (a) or (b) is introduced optical grating, which is oriented perpendicular to the path of an excitation light, which is coupled by means of the optical grating in the thin-film waveguide,
c. auf der Oberfläche des Dünnschichtwellenleiters ein Immunoassay in Form einer Substanzbibliothek von immobilisierten Bindungspartnern für Analyten und/oder für Analytensonden in Reihen von räumlich getrennten Messbereichen und ein unabhängiger Kontroll-Assay beinhaltend das Referenzierungsantigen immobilisiert in Reihen von räumlich getrennten Messbereichen aufgetragen sind, undc. on the surface of the thin-film waveguide, an immunoassay in the form of a substance library of immobilized binding partners for analytes and / or analyte probes in rows of spatially separated measurement areas and an independent control assay comprising the homing antigen immobilized in rows of spatially separated ranges are plotted, and
d. die jeweiligen Reihe parallel zum optischen Gitter orientiert sind und sich in der Richtung des Anregungslichtes oberhalb und unterhalb jeder Reihe des Immunoassays eine Reihe von Kontroll-Assays befindet.d. the respective rows are oriented parallel to the optical grating and there is a series of control assays in the direction of the excitation light above and below each row of the immunoassay.
2. Kartusche nach Anspruch 1, dadurch gekennzeichnet, dass das Referenzierungsantigen ein ähnliches Molekulargewicht wie das des Analyten hat, die Referenzierungssonde ähnliche Bindungseigen- schatten wie die Analytensonden aufweist, der Kontroll-Assay keine Kreuzreaktivität zu den Immunoassays zeigt und das Referenzierungsantigen in der untersuchten Matrix nicht vorkommt.2. Cartridge according to claim 1, characterized in that the homing antigen has a molecular weight similar to that of the analyte, the homing probe has similar binding properties as the analyte probes, the control assay shows no cross-reactivity with the immunoassays and the homing antigen in the examined matrix does not occur.
3. Kartusche nach einem der Ansprüche 1 oder 2, wobei die Analytensonden Antikörper sind.3. A cartridge according to any one of claims 1 or 2, wherein the analyte probes are antibodies.
4. Kartusche nach Anspruch 1 bis 3, dadurch gekennzeichnet, dass die Analyten Mykotoxine sind.4. A cartridge according to claim 1 to 3, characterized in that the analytes are mycotoxins.
5. Kartusche nach Anspruch 4, wobei in dem Kontroll-Assay das Referenzierungsantigen < 1000 g/mol ist.A cartridge according to claim 4, wherein in the control assay the referencing antigen is <1000 g / mol.
6. Kartusche nach einem der Ansprüche 4 oder 5, wobei das Referenzierungsantigen Fluorescein ist. - -A cartridge according to any one of claims 4 or 5, wherein the referencing antigen is fluorescein. - -
7. Kartusche nach einem der Ansprüche 4 bis 6, wobei das Immunoassay Mykotoxin-Protein- Konjugaten und / oder das Kontroll-Assay Kontrollmolekül-Protein-Konjugaten enthalten.A cartridge according to any one of claims 4 to 6, wherein the immunoassay contains mycotoxin-protein conjugates and / or the control assay comprises control-protein conjugates.
8. Verfahren zur quantitativen Analyse von Analyten, das folgende Schritte umfasst:8. A method for the quantitative analysis of analytes comprising the steps of:
a. optionale Extraktion der Analyten aus einer Matrix in eine Probeflüssigkeit,a. optional extraction of the analytes from a matrix into a sample liquid,
b. Durchführung des Assays in der Kartusche nach einem der Ansprüche 1 bis 7, wobei nachb. Carrying out the assay in the cartridge according to one of claims 1 to 7, wherein
Einführung der Probeflüssigkeit in die Kartusche, die Probeflüssigkeit in die Reagenzienkammer befördert wird und sich mit dort aufgebrachten markierten Sonden vermischt bzw. reagiert, dannIntroduce the sample liquid into the cartridge, transport the sample fluid into the reagent chamber and mix or react with labeled probes applied there, then
c. Beförderung der Probeflüssigkeit in die Detektionskammer und Reaktion der Analyten und/oder die markierten Sonde mit dem Immunoassay und Kontroll-Assay, gefolgt vonc. Transport of the sample liquid into the detection chamber and reaction of the analytes and / or the labeled probe with the immunoassay and control assay, followed by
d. Ausleuchtung des Dünnschichtwellenleiters zur Anregung der markierten Sonden des Im- munoassays und Kontroll-Assays zur Fluoreszenz und Aufnahme eines Fluoreszenzbildes, dannd. Illumination of the thin-film waveguide for excitation of the labeled probes of the immunoassay and control assay for fluorescence and recording of a fluorescence image, then
e. Berechnung der referenzierten Fluoreszenzintensitäten des Immunoassays anhand des Kon- troll-Assays, wobei die referenzierte Fluoreszenzintensität eines jeden Immunoassay-e. Calculation of the referenced fluorescence intensities of the immunoassay by means of the control assay, wherein the referenced fluorescence intensity of each immunoassay
Messbereiches durch Division der Fluoreszenzintensität des Immunoassay-Messbereiches durch den Mittelwert der Fluoreszenzintensitäten der in der Richtung des Anregungslichtes benachbarten Kontroll-Assay-Messbereiche berechnet wird, undMeasuring range is calculated by dividing the fluorescence intensity of the immunoassay measuring range by the mean value of the fluorescence intensities of the adjacent in the direction of the excitation light control assay ranges, and
f. Berechnung und Anzeige der Analyten- Werte durch Bezug auf eine Kalibrationskurve.f. Calculation and display of the analyte values by reference to a calibration curve.
9. Verfahren nach Anspruch 8, bei dem die Kartusche über die Dauer des Verfahrens auf eine Temperatur von 20 bis 37 0C temperiert wird.9. The method of claim 8, wherein the cartridge is tempered over the duration of the process to a temperature of 20 to 37 0 C.
10. Verfahren nach einem der Ansprüche 8 oder 9, wobei die Dauer der Reaktion im Schritt b. 1 bis 20 min beträgt und/oder die Dauer der Reaktion im Schritt c. Ibis 100 min beträgt.10. The method according to any one of claims 8 or 9, wherein the duration of the reaction in step b. 1 to 20 minutes and / or the duration of the reaction in step c. Ibis is 100 min.
11. Verwendung der Kartusche nach einem der Ansprüche 1 bis 7 und des Verfahrens nach einem der Ansprüche 8 bis 10 zum Nachweis und zur quantitativen Analyse von Mykotoxinen. 11. Use of the cartridge according to one of claims 1 to 7 and the method according to one of claims 8 to 10 for the detection and quantitative analysis of mycotoxins.
EP10712339A 2009-04-09 2010-03-26 Device and method for the verification and quantitative analysis of analytes, particularly mycotoxins Withdrawn EP2417436A1 (en)

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CR20110530A (en) 2012-01-31
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CN102460127A (en) 2012-05-16
CL2011002509A1 (en) 2012-04-20
BRPI1015212A2 (en) 2016-05-03
WO2010115530A1 (en) 2010-10-14
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US20130203613A1 (en) 2013-08-08
KR20120014122A (en) 2012-02-16

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