Classifications

• • 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
  • 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
• • 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
  • 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
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
• • 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
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of 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
  • G01N21/75—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
  • 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
  • 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
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/02—Food
• • 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
  • G01N33/02—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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