EP1287360A2 - Kit et procede pour la detection d'une pluralite d'analytes - Google Patents

Kit et procede pour la detection d'une pluralite d'analytes

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Publication number
EP1287360A2
EP1287360A2 EP01940527A EP01940527A EP1287360A2 EP 1287360 A2 EP1287360 A2 EP 1287360A2 EP 01940527 A EP01940527 A EP 01940527A EP 01940527 A EP01940527 A EP 01940527A EP 1287360 A2 EP1287360 A2 EP 1287360A2
Authority
EP
European Patent Office
Prior art keywords
luminescence
kit according
analytes
sensor platform
layer
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Withdrawn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
EP01940527A
Other languages
German (de)
English (en)
Inventor
Michael Pawlak
Eginhard Schick
Andreas P. Abel
Gert L. Duveneck
Markus Ehrat
Gerhard M. Kresbach
Eveline SCHÜRMANN-MADER
Martin A. Bopp
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
Zeptosens 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 Zeptosens AG filed Critical Zeptosens AG
Publication of EP1287360A2 publication Critical patent/EP1287360A2/fr
Withdrawn legal-status Critical Current

Links

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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell 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
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • 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
    • 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"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • 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
    • G01N2021/6482—Sample cells, 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
    • G01N21/62—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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/808—Optical sensing apparatus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/808—Automated or kit

Definitions

  • the invention relates to various embodiments of a kit for the simultaneous qualitative and / or quantitative detection of a large number of analytes.
  • the invention also relates to analytical systems based on the kit according to the invention and methods carried out therewith for the detection of one or more analytes and their use.
  • microtiter plates For the determination of a large number of analytes, above all methods are widespread in which the detection of different analytes in so-called microtiter plates is carried out in discrete sample containers or "wells" of these plates.
  • the most widespread are plates with a grid of 8 x 12 wells on a base area of typically approx. 8 cm x 12 cm, a volume of a few hundred microliters being required to fill an individual well.
  • US Pat. No. 5,747,274 describes measurement arrangements and methods for the early detection of a heart attack by the determination of a plurality of at least three heart attack markers, the determination of these markers being able to take place in individual or in a common sample container, in the latter case following the description given, a single sample container is designed as a continuous flow channel, the boundary surface of which, for example, forms a membrane on which antibodies for the three different markers are immobilized.
  • a single sample container is designed as a continuous flow channel, the boundary surface of which, for example, forms a membrane on which antibodies for the three different markers are immobilized.
  • no geometrical information about the size of the measuring areas is given.
  • the immobilization for the analyte-specific recognition elements is in the form of small "spots" with partially well under 1 mm 2 area on solid supports proposed in order to be able to carry out a concentration determination of the analyte which is dependent only on the incubation time but - in the absence of a continuous flow - essentially independent of the absolute sample volume by binding only a small part of the analyte molecules present.
  • the measurement arrangements described in the associated exemplary embodiments are based on the detection of ruorescence in conventional microtiter plates.
  • a light wave is coupled into an optical waveguide which is surrounded by optically thinner media, ie media with a lower refractive index, it is guided by total reflection at the interfaces of the wave-guiding layer.
  • a fraction of the electromagnetic energy enters the optically thinner media. This proportion is known as the evanescent or cross-damped field.
  • the strength of the evanescent field is very much dependent on the thickness of the waveguiding layer itself and on the ratio of the refractive indices of the waveguiding layer and the media surrounding it.
  • the first proposed measuring arrangements of this type were based on highly multimodal, self-supporting single-layer waveguides, such as fibers or platelets made of transparent plastic or glass, with thicknesses from a few hundred micrometers to several millimeters.
  • Planar thin-film waveguides have been proposed to improve sensitivity and, at the same time, simplify mass production.
  • a planar thin-film waveguide consists of a three-layer system: carrier material, waveguiding layer, superstrate (or sample to be examined), the waveguiding layer having the highest refractive index. Additional intermediate layers can improve the effect of the planar waveguide.
  • luminescence denotes the spontaneous emission of photons in the ultraviolet to infrared range after optical or non-optical, such as, for example, electrical or chemical or biochemical or thermal excitation.
  • luminescence-based methods appear to be more suitable than methods based on a change in the effective refractive index (such as grating coupler sensors or methods based on surface plasmon resonance) due to the greater selectivity of signal generation.
  • the luminescence excitation is limited to the depth of penetration of the evanescent field into the optically thinner medium, that is to say to the immediate vicinity of the wave-guiding region with a depth of penetration into the medium of the order of magnitude of a few hundred nanometers. This principle is called evanescent luminescence excitation.
  • WO 95/33197 describes a method in which the excitation light is coupled into the waveguiding film as a diffractive optical element via a relief grating.
  • the surface of the sensor platform is brought into contact with a sample containing the analyte, and the isotropically emitted luminescence in the penetration depth of the evanescent field of luminescent substances is measured by means of suitable measuring devices, such as photodiodes, photomultipliers or CCD cameras. It is also possible to decouple and measure the portion of the evanescently excited radiation fed back into the waveguide via a diffractive optical element, for example a grating. This method is described for example in WO 95/33198.
  • spatially separated measuring ranges or “discrete measuring ranges” in the sense of the present invention is defined in more detail in the later section for describing the present invention.
  • WO 98/22799 also proposes devices which correspond to the shape of known microtiter plates.
  • US 5525466 and US 5738992 describe an optical sensor based on fluorescence excitation in the evanescent field of a self-supporting multimode waveguide, preferably of a fiber-optic type. The excitation light is coupled in and the fluorescent light fed back into the multimode waveguide is coupled out via coupling in and out of the end face.
  • the fluorescence signal detected in the process for the analyte detection results from the functional principle of such multimode waveguides as a single integral value for the entire surface interacting with the sample.
  • fluorescent reference materials are co-immobilized on the sensor surface in addition to the biochemical or biological detection elements for the specific detection and binding of an analyte to be detected. Due to the underlying sensor principle, however, it is not possible to have a spatially resolved, but only a normalization that affects the individual, integral measured value. Consequently, the detection of different analytes can only be carried out by using lab in different excitation wavelengths or sequentially, after removal of previously bound analytes. For these reasons, these arrangements, together with the referencing method described, do not appear to be suitable, or only at all, for the simultaneous detection of a large number of analytes.
  • EP-A-093613 describes a method for referencing in the area adjacent to the "measuring range". In particular, this emphasizes the need to use reference and measurement signals from the same areas (on the sensor) on a sensor platform.
  • Kinetic (time-resolved) measurements are mentioned as a possible implementation, since the kinetics of the analyte binding are not dependent on the physical waveguide parameters and possible defects which affect the signals locally impact.
  • a disadvantage of the kinetic method is its dependence on external parameters such as temperature and viscosity of the individual sample. No.
  • 5631170 describes the referencing by means of co-immobilized fluorophores which generate a reference signal which is independent of the analyte concentration. It is preferred that the specific recognition elements for analyte binding and the co-immobilized fluorophores used for referencing are present in a statistical mixture on the sensor platform. Furthermore, based on the exemplary embodiment of a "capillary fill device" (CFD), a method for simultaneous calibration is presented by using known amounts of the analyte in addition to the sample (for example in a competitive assay format) in local zones of the CFD, for example by they are released by adding the sample (for example opposite the sensor surface) to the designated reagent containers.
  • CFD capillary fill device
  • WO 97/35181 describes methods for the simultaneous determination of one or more analytes by applying patches with different detection elements in a "well" formed in the waveguide, which patches are contacted with a sample solution containing one or more analytes.
  • solutions with defined analyte concentrations are simultaneously added to other wells with similar patches.
  • 3 wells for measurement with calibration solutions of low and high analyte concentration and the current sample
  • discrete and immobilized detection elements that differ from patch to patch are presented for the simultaneous determination of several analytes. There are no indications of spatially resolved referencing.
  • the sandwich immunoassays are carried out with sequential addition of washing solution (buffer), sample with one or more analytes, washing solution (buffer), tracer antibody (individually or as a cocktail) and washing solution (buffer).
  • the locally measured fluorescence intensities are corrected by subtracting the background signal observed next to the measuring fields.
  • this arrangement also does not make it possible to carry out an entire series of measurements for the simultaneous determination of several analytes, together with the necessary calibrations, but instead requires either the use of several different sensor platforms or repetitive, sequential measurements on a platform with interim regeneration, which is particularly the case with In many cases immunoassays are only possible to a limited extent.
  • the invention relates to a kit for the simultaneous qualitative and / or quantitative detection of a large number of analytes, comprising - A sensor platform comprising an optical thin-film waveguide with a layer (a) which is transparent at at least one excitation wavelength, on a layer (b) which is likewise transparent at at least this excitation wavelength and which has a lower refractive index than layer (a) and at least one grating structure modulated in layer (a) ( c) for coupling said excitation light into layer (a),
  • kit according to the invention it was found that, using a kit according to the invention, it is possible in multianalyt assays to simultaneously determine several analytes in a sample to achieve a sensitivity and reproducibility which is similar to that in a corresponding number of individual assays for the detection of individual analytes.
  • spatially separated or discrete measurement areas are to be defined by the closed area, the biological or biochemical or synthetic recognition elements immobilized there for recognition of an analyte from a liquid sample.
  • These surfaces can have any geometry, for example the shape of points, circles, rectangles, triangles, ellipses or lines.
  • optical transparency is understood below to mean that the material characterized by this property should be largely transparent and thus absorption-free at least at one or more excitation wavelengths used to excite one or more luminescence.
  • the sensitivity of an arrangement according to the invention is greater, the higher the difference between the refractive index of layer (a) and the refractive index of the surrounding media, i.e. the higher the refractive index of layer (a). It is preferred that the refractive index of the first optically transparent layer (a) is greater than 1.8.
  • the first optically transparent layer (a) is a material from the group of TiO 2 , ZnO, Nb 2 O 5 , Ta 2 O 5 , HfO 2 , or ZrO 2 , particularly preferably made of TiO 2 or Ta 2 O 5 or Nb 2 Ü 5 . Combinations of several such materials can also be used.
  • the sensitivity up to a lower limit of the layer thickness is greater, the smaller the layer thickness.
  • the lower limit is determined by the termination of the light guide when the value falls below a value which depends on the wavelength of the light to be guided, and an observed increase in the propagation losses in the case of very thin layers with a further decrease in layer thickness. It is advantageous if the product of the thickness of the layer (a) and its refractive index is one tenth to one Whole, preferably one third to two thirds, of the excitation length of an excitation light to be coupled into the layer (a).
  • the optically transparent layer (b) should be low in absorption and fluorescence, ideally free of absorption and fluorescence.
  • the surface roughness should be low, since the surface roughness of the layer (b) upon deposition of a further layer (a) with a higher refractive index, which is intended as a waveguiding layer, depends to a greater or lesser extent on the surface roughness of the layer, depending on the deposition process (a) affects.
  • An increased surface roughness at the boundary layers of layer (a) leads to increased scatter losses of the guided light, but this is undesirable.
  • the material of the second optically transparent layer (b) contains silicates, e.g. B. glass or quartz, or a transparent thermoplastic or sprayable plastic, for example from the group comprising polycarbonate, polyimide, acrylate, in particular polymethyl methacrylate, or polystyrene.
  • lattice structures (c) modulated in layer (a) have a period of 200 nm - 1000 nm and their modulation depth is 3 to 100 nm, preferably 10 to 50 nm. It is preferred that the ratio of the modulation depth to the thickness of the first optically transparent layer (a) is equal to or less than 0.4.
  • the lattice structure can be designed in various forms. It is preferred that the grating structure (c) is a relief grating with any profile, for example with a rectangular, triangular or semicircular profile, or a phase or volume grating with a periodic modulation of the refractive index in the essentially planar optically transparent layer (a) is.
  • the lattice structure (c) is a diffractive lattice with a uniform period.
  • the grating structure (c) is a multi-diffractive grating.
  • the grating structure (c) has a periodicity which varies spatially perpendicular or parallel to the direction of propagation of the excitation light coupled into the optically transparent layer (a).
  • the sensor platform comprises uniform, unmodulated regions of the layer (a), which are preferably arranged in the direction of propagation of the excitation light coupled in via a grating structure (c) and guided in the layer (a).
  • lattice structures (c) can be used to couple excitation light to the measurement areas (d) and / or to couple luminescence grown back into layer (a).
  • the sensor platform will therefore comprise a plurality of lattice structures (c) of the same or different period with optionally adjoining uniform, unmodulated regions of the layer (a) on a common, continuous substrate.
  • a grating structure (c) to which an unmodulated area of the layer (a) is directed in the direction of propagation of the light that is coupled in and guided in the layer (a).
  • a large number of measuring ranges in an array on which the different analytes are detected.
  • this will advantageously be followed by a further lattice structure with a further array of measurement areas located behind it, etc. After passing through an unmodulated area, the light guided in layer (a) is coupled out again.
  • each array of measurement areas following in the direction of propagation of the coupled excitation light is assigned a grating structure (c) specific to this array for decoupling this excitation light, wherein the grating structures can be formed perpendicular to the direction of propagation of the coupled excitation light or can also be formed specifically for individual arrays can extend over the entire sensor platform in this direction.
  • the coupling-in grating of an array following in the direction of propagation of an excitation light guided in the layer (a) of a sensor platform serves as coupling-out grating for the excitation light coupled in on the coupling-in grating of the array preceding in said direction of propagation.
  • the sensor platform comprises an overlay of 2 or more grating structures of different periodicity with parallel or non-parallel, preferably non-parallel, alignment of the grating lines, which serves to couple excitation light of different wavelengths, in the case of two superimposed grating structures, the grating lines of which are preferably oriented perpendicular to one another.
  • the subdivision of the sensor platform into areas with grating structures and adjoining unmodulated areas means that the space required for a single array of measuring areas between successive grating structures (including at least one assigned grating structure) cannot fall below a certain minimum, which the current technical possibilities for generating the lattice structures and for coupling in a suitable excitation light bundle in the order of magnitude
  • 9 9 is from about 0.1 mm to 1 mm. It is therefore particularly advantageous for arrangements in which a large number of small-area arrays is desired if a lattice structure (c) or a superposition of a plurality of lattice structures in layer (a) is modulated essentially over the entire area of the sensor platform.
  • optically or mechanically recognizable markings are applied to the sensor platform to facilitate adjustment in an optical system and / or for connection to sample containers as part of an analytical system.
  • a further embodiment of the arrangement according to the invention is that between the optically transparent layers (a) and (b) and in contact with layer (a) there is another optically transparent layer (b ') with a lower refractive index than that of layer (a ) and a thickness of 5 nm - 10000 nm, preferably of 10 nm - 1000 nm.
  • the simplest form of immobilization of the biological or biochemical or synthetic recognition elements consists in physical adsorption, for example as a result of hydrophobic interactions between the recognition elements and the base plate.
  • these interactions can be greatly changed in their extent by the composition of the medium and its physicochemical properties, such as polarity and ionic strength.
  • the adhesion of the recognition elements after purely adsorptive immobilization on the surface is often inadequate.
  • the adhesiveness is improved in that an adhesion-promoting layer (f) is applied to the sensor platform for immobilizing biological or biochemical or synthetic recognition elements.
  • the adhesion promoter layer can also serve, in particular in the case of biological or biochemical recognition elements to be immobilized, to improve the "biocompatibility", ie to maintain the binding capacity compared to its extent in a natural way biological or biochemical environment, and in particular avoiding denaturation. It is preferred that the adhesion promoting layer (f) has a thickness of less than 200 nm, preferably less than 20 nm. A large number of materials are suitable for producing the adhesion-promoting layer. Without any limitation, it is preferred that the adhesion promoting layer (f) comprise one or more chemical compounds from the groups comprising silanes, epoxies, functionalized, charged or polar polymers and "self-organized passive or functionalized mono- or double layers".
  • kits according to the invention are immobilized in spatially separated measuring areas (d). These spatially separated measuring areas (d) can be generated by spatially selective application of biological or biochemical or synthetic detection elements on the sensor platform.
  • a large number of known processes are suitable for the application. Without restricting generality, it is preferred that one or more methods from the group of methods used by "ink jet spotting”, mechanical spotting by means of pen, pen or capillary are used to apply the biological or biochemical or synthetic recognition elements to the sensor platform.
  • “Micro contact printing” fluidic contacting of the measuring areas with the biological or biochemical or synthetic recognition elements by their supply in parallel or crossed microchannels, under the influence of pressure differences or electrical or electromagnetic potentials, as well as photochemical and photolithographic immobilization processes.
  • components from the group can be applied which consist of nucleic acids (for example DNA, RNA, OUgonukelotiden) or nucleic acid analogs (for example PNA), mono- or polyclonal antibodies, aptamers, synthetic peptide structures, soluble, membrane-bound and proteins isolated from a membrane, such as receptors, their ligands, antigens for antibodies, "histidine tag Components "and their complex formation partners, cavities generated by chemical synthesis for receiving molecular imprints. It is also provided that whole cells, cell components, cell membranes or their fragments are applied as biological or biochemical or synthetic recognition elements.
  • kits according to the invention are that the density of the detection elements immobilized in discrete measurement areas for the detection of different analytes on different measurement areas is selected such that the luminescence signals when detecting different analytes in a common array are of the same order of magnitude, that is to say that the The associated calibration curves for the analyte determinations to be carried out simultaneously can be recorded without changing the optoelectronic system settings.
  • arrays of measuring ranges are divided into segments of one or more measuring ranges for determining analytes and measuring ranges for referencing, i.e. Determination of physical parameters and / or chemical differences between different applied samples.
  • one or more arrays can comprise segments of two or more measurement areas with biological or biochemical or synthetic recognition elements of the same type within the segment for analyte determination or referencing.
  • a segment can also contain several discrete measurement areas with different detection elements.
  • a possible embodiment of the kit according to the invention is characterized in that one or more segments of an array or one or more arrays are assigned to the determination of analytes from a common group, such as with immobilized anti-cytokine antibodies for the determination of different cytokines.
  • one or more segments of an array or one or more arrays can serve for the simultaneous determination of an entire set of so-called "marker proteins”. For example, this intra- and / or extracellularly occurring proteins of the body, which occur as a result of and as signs of various diseases, such as degenerative diseases, certain forms of cancer or autoimmune diseases, for example in an increased concentration.
  • a recognition element to be immobilized for the detection of an analyte is selected such that it has the highest possible specificity and binding affinity for the recognition and binding of said analyte and as little cross-reactivity as possible with other, possibly (bio) chemically similar analytes.
  • cross-reactivity with the (bio) chemically most similar relatives of the analyte in question can hardly be ruled out.
  • kit according to the invention can be advantageous for such applications, in which one or more measurement areas of a segment or an array are assigned to the determination of the same analyte and whose immobilized biological or biochemical recognition elements have different affinities for said analyte.
  • the recognition elements are expediently selected such that their affinities for different, (bio) chemically similar analytes change in different, characteristic ways.
  • the identity of the analyte can then be determined from the entirety of the signals from different measurement areas with different detection elements for a single analyte, in a manner comparable to a fingerprint.
  • Another embodiment is characterized in that one or more segments of an array or one or more arrays of the determination of different groups of analytes, such as pharmaceutical preparations ("drugs") for the treatment of a disease and / or its metabolites and / or the detection substances for this disease, such as so-called “marker proteins”, are assigned.
  • This makes it possible to determine, in a single measurement, the concentration of a whole set of "marker proteins", as explained above, and the concentration of preparations administered for the treatment of a disease and of their breakdown products.
  • two or more arrays it is advantageous for two or more arrays to have a similar geometric arrangement of measurement areas and / or segments of measurement areas for the determination of similar ones Have analytes on these arrays.
  • the kit according to the invention with a large number of measuring ranges in discrete arrays, a large number of which in turn can be arranged on a common sensor platform offers the possibility of using relatively small amounts of sample solutions, reagents or, if appropriate, calibration solutions on one and the same platform, under largely identical conditions, many types of duplications or multiple executions of the same measurements can be carried out. In this way, for example, statistical data can be generated in a single measurement, for which a large number of individual measurements with a correspondingly longer total measurement time and higher consumption of sample and reagent quantities is conventionally required. It is preferred that 2 or more for each analyte or for physical or chemical referencing identical measuring ranges are provided within a segment or array.
  • said identical measurement areas can be arranged in a continuous row or column or diagonals of an array or segment of measurement areas.
  • the referencing aspects can relate to physical or chemical parameters of the sensor platform, such as local differences in the excitation light intensity (see also below), as well as influences of the sample, such as its pH, ionic strength, refractive index, temperature etc.
  • said identical measuring ranges are arranged statistically within an array or segment of measuring ranges.
  • the immobilized recognition elements are generally selected so that they recognize and bind the analyte to be detected with the highest possible specificity. In general, however, it is to be expected that non-specific attachment of analyte molecules to the surface of the base plate also takes place, in particular if reactive clearances are still present between the detection elements immobilized in the measurement areas.
  • areas between the spatially separated measuring areas are "passivated” to minimize non-specific binding of analytes or their detection substances, ie that "chemically neutral” compounds are applied between the spatially separated measuring areas (d) with respect to the analyte, preferably, for example, existing from the groups which contain albumin, in particular bovine serum albumin or human serum albumin, casein, nonspecific, polyclonal or monoclonal, non-specific or empirically unspecific antibodies for the analyte (s) to be detected (especially for immunoassays), detergents such as Tween 20 Polynucleotides of hybridizing, fragmented natural or synthetic DNA, such as an extract of herring or salmon sperm (especially for polynucleotide hybridization assays), or also uncharged but hydrophilic polymers, such as polyethylene glycols or dextrans.
  • albumin in particular bovine serum albumin or human serum albumin, casein, nonspecific, polyclonal or monoclonal, non-specific or empirical
  • kits according to the invention are advantageous for many, if not the majority of applications, in which an adhesion-promoting layer was applied to the sensor platform before the immobilization of the biological or biochemical or synthetic detection elements.
  • Preferred embodiments are those which are characterized in that the function of passivating areas between the spatially separated measuring areas to minimize non-specific binding of analytes or their detection substances is carried out by applying said adhesion-promoting layer on the sensor platform without applying additional substances.
  • the kit according to the invention can comprise a very large number of individual measuring ranges. It is preferred that up to 100,000 measuring ranges are arranged in a 2-dimensional arrangement and that a single measuring range takes up an area of 0.001-6 mm 2 .
  • Another object of the invention is an embodiment of the kit according to the invention, in which the upper side of the sensor platform with the measurement areas generated thereon is brought together with another body above the optically transparent layer (a) in such a way that one or more between the sensor platform as the base plate and said body spatial cutouts for generating one or more sample containers fluidly sealed against one another are produced, in each of which there are one or more measuring areas or segments or arrays of measuring areas.
  • the body to be brought together with the sensor platform is understood not only to be self-supporting structures, but also, for example, applied structured coatings of possibly only a few micrometers in thickness, which, under the conditions of use of the kit, cause liquid to pass through in such a way (in this case prevent typically open) sample container into an adjacent sample container.
  • the sample containers are designed as flow cells that are fluidically sealed from one another, each with at least one inlet and at least one outlet and, if appropriate, additionally leads at least one outlet of each flow cell into a reservoir that is fluidly connected to this flow cell and contains liquid that emerges from the flow cell receives.
  • the optionally additionally available reservoir for receiving liquid exiting the nut cell is designed as a depression in the outer wall of the body brought together with the sensor platform as the base plate.
  • spatial structures in the grid of the array of the soot cells to be produced are formed on the sensor platform as the base plate.
  • These structures on the base plate can form, for example, the walls or parts of the walls, such as bases, between the soot cells arranged next to and behind one another, which are produced by bringing the base plate together with a correspondingly shaped body.
  • recesses it is also possible for recesses to be formed in the sensor platform in order to generate the spatial cutouts between the sensor platform as the base plate and the body brought together therewith.
  • a further embodiment consists in that recesses are formed in said body in order to produce the recesses between the base plate and the body brought together therewith. It is preferred for this embodiment that the base plate is essentially planar.
  • the body to be brought together with the base plate for producing the array of soot cells can consist of a single workpiece.
  • the body brought together with the base plate consists of several Parts is assembled, the assembled components of said body preferably forming an irreversibly assembled unit.
  • the body brought together with the base plate comprise auxiliary means which facilitate the assembly of said body and the base plate.
  • the arrangement preferably comprises a plurality, i. H. 2 - 2000 sample containers, preferably 2 - 400, particularly preferably 2 - 100 sample containers.
  • sample containers are open on the side of the body which is brought together with the sensor platform as the base plate and is opposite the measurement areas.
  • the grid (sequence in rows and / or columns) of the sample containers corresponds to the grid of the wells of a standard microtiter plate.
  • Another embodiment of the arrangement of sample containers as part of the kit according to the invention is characterized in that it is closed by an additional closure, for example a film, membrane or a cover plate.
  • the absorption capacity of the soot cells can be varied within a wide range, so that the internal volume of each sample container is typically 0.1 ⁇ l - 1000 ⁇ l, preferably 1 ⁇ l - 20 ⁇ l.
  • the internal volumes of different soot cells of an arrangement can be the same or different.
  • the depth of the recesses between the sensor platform as the base plate and the body joined therewith is 1 to 1000 ⁇ m, particularly preferably 20 to 200 ⁇ m.
  • the size of the recesses in an array can be be uniform or different, and the base areas can have any, preferably rectangular or polygonal or other geometry.
  • the lateral dimensions of the base areas can be varied within a wide range, typically the base areas of the recesses between the base plate and the body joined therewith each being 0.1 mm 2 - 200 mm 2 , preferably 1 mm 2 - 100 mm 2 .
  • the corners of the bases are rounded. Rounded corners have a favorable effect on the flow profile and facilitate the removal of any gas bubbles that may have formed from the soot cells or prevent their formation.
  • multi-channel pipettors can be used for manual or automatic reagent application, in which the individual pipettes are arranged in one- or two-dimensional arrays, provided that the arrangement of sample containers as part of the kit according to the invention includes the inlets in the has the appropriate grid.
  • the grid (sequence in rows and columns) of the arrangement therefore preferably corresponds to the grid of the wells of standard microtiter plates.
  • An arrangement of 8 x 12 wells with a (center-to-center) spacing of approx. 9 mm has been established as an industrial standard. Smaller arrays with 3, 6, 12, 24 and 48 wells are equally compatible with this. It is also possible to combine several arrangements of sample containers according to the invention with such smaller arrays of flow cells in such a way that the individual inlets of said soot cells are arranged in an integral multiple of the spacing of approximately 9 mm.
  • plates with 384 and 1536 wells, an integral multiple of 96 wells on the same footprint with a correspondingly reduced well spacing, have also been used, which are also to be referred to as standard microtiter plates.
  • standard microtiter plates By adapting the grid of the sample containers of the arrangement according to the invention, with the inlets and outlets of each sample container these standards can be used with a variety of commercially available and available laboratory pipettors and robots for sample addition.
  • the outer basic dimensions of the arrangement of sample containers, as part of the kit according to the invention preferably correspond to the basic dimensions of these standard microtiter plates.
  • Another special form of the invention is an arrangement of, for example, 2 to 8 sample containers as part of the kit according to the invention, with the properties mentioned above, in a column or, for example, 2 to 12 sample containers in a row, which on the other side is supported with a carrier ("meta carrier").
  • a carrier metal carrier
  • the assembly of the sample containers with the meta carrier can be done, for example, by gluing or by exact adjustment without gluing if it is intended for single use, or for example by latching or inserting it into a suitably designed holder if it is intended for multiple use.
  • the material of the meta carrier can, for example, be selected from the group consisting of moldable, sprayable or millable plastics, metals, silicates, such as glass, quartz or ceramics
  • sample containers with a single meta carrier in such a way that the grid (sequence in rows or columns) of the inflows of the soot cells corresponds to the grid of the wells of a standard microtiter plate, i.e. an integer multiple of 9 mm (corresponding to 96 -Well plate) or of 4.5 mm (corresponding to 384-well plate, see above) or of 2.25 mm (corresponding to 1536-well plate, see above).
  • the arrangement of sample containers can of course also be designed in a different grid.
  • the materials for the body brought together with the sensor platform as the base plate and any additional cover plate that may be used must meet the requirements for the planned use of the arrangement. Depending on the specific application, these requirements relate to chemical and physical resistance, for example against acidic or basic media, salts, alcohols or detergents as components of aqueous solutions, or formamide, temperature resistance (for example between -30 ° C and 100 ° C) , possible similar thermal expansion coefficients of the base plate and the body brought together, optical properties (e.g. freedom from ruorescence, reflectivity), mechanical workability etc. It is preferred that the material of the body brought together with the base plate and an optional additional closure from the same group as the material of the "meta carrier" is selected.
  • the components mentioned (body combined with the sensor platform as the base plate, cover plate) can each consist of a uniform material and also comprise a mixture or layer-by-layer or lateral connection of different materials, the materials being able to replace one another.
  • An extremely essential aspect of the present invention is the possibility of spatially resolved referencing of the available excitation light intensity.
  • the available excitation light intensities of an illuminated area are essentially determined by the excitation light density in the cross section of the excitation light bundle.
  • Local variations in the properties of the illuminated areas (such as a glass plate) have only a secondary influence here.
  • there are local variations in the physical parameters of the sensor platform such as the coupling efficiency of the grating structure (c) for coupling the excitation light into the optically transparent layer (a), or local variations in the propagation losses guided modes in the optically transparent layer (a), of crucial importance.
  • kits according to the invention which are characterized in that the precautions for spatially resolved referencing of the excitation light intensity available in the measurement ranges include the simultaneous or sequential generation of an image of the light emitted by the sensor platform at the excitation wavelength. It is assumed that the scattered light losses are essentially proportional to the locally guided light intensity. The scattered light losses are mainly determined by the surface roughness and homogeneity of the optically transparent layer (a) and the substrate located below (optically transparent layer (b)).
  • this type of referencing enables a reduction in the locally available excitation light intensity in its direction of propagation to be taken into account if this was done, for example, by absorption of excitation light by a high local concentration in the evanescent field of layer (a) of molecules which are absorbing at the excitation wavelength.
  • the assumption of the proportionality of the emitted scattered light to the intensity of the guided light does not apply in those places where radiation / decoupling takes place through local macroscopic scattering centers that are in contact with layer (a). At these points, the emitted scattered light is significantly disproportionate to the guided light. It is therefore also advantageous if the precautions for spatially resolved referencing of the excitation light intensity available in the measurement areas include the simultaneous or sequential generation of an image of the light emitted by the sensor platform at the luminescence wavelength. Both methods can of course also be combined with one another. When creating a reference image, different influences of the imaging optics on the acquisition of the measurement signals should be excluded.
  • the image of the excitation light emitted by the sensor platform is created via the same optical path as the detection of the luminescence originating from the measurement areas.
  • the precautions for spatially resolved referencing of the excitation light intensity available in the measurement areas include the simultaneous or sequential creation of an image of the light emitted by the sensor platform at an excitation wavelength other than for excitation of a luminescence. It is preferred that an excitation wavelength is selected in which luminescent molecules used in the course of the method for the detection of one or more analytes or for the purposes of referencing or calibration have no absorption or only the lowest possible absorption, so that effects of "photochemical bleaching" can be avoided or minimized.
  • the spatial resolution of the image for referencing the excitation light emitted by the sensor platform on the sensor platform is better than 100 ⁇ m, preferably better than 20 ⁇ m. It is also preferred that the precautions for spatially resolved referencing of the excitation light intensity available in the measurement areas include determining the background signal at the respective luminescence wavelength next to or between the measurement areas.
  • a preferred embodiment of the kit according to the invention is characterized in that the spatially resolved referencing of the excitation light intensity available in the measuring ranges by means of "luminescence marker spots", i.e.
  • the luminescence intensity is determined from measurement areas with pre-immobilized (i.e., already applied in these measurement areas before adding a sample) luminescence-labeled molecules. It is preferred that the “luminescence marker spots” are applied in a grid that spans the entire sensor platform.
  • position-resolving detectors such as CCD cameras
  • CCD cameras are preferably used for the signal detection.
  • These are characterized by the fact that their photosensitive elements (pixels) have a specific (especially thermal) background signal, which is the lower threshold of the Detection of a local light signal determined, and also have a maximum capacity (saturation) for detection of high light intensities.
  • the difference between these threshold values determines the dynamic range of the signal detection for a given exposure time.
  • Both the luminescence signals to be detected for analyte detection and the reference signals should move within this dynamic range. It is advantageous if both signals are of the same order of magnitude, ie differ by no more than one or two powers of ten, for example.
  • this can be achieved, for example, by selecting the density of the luminescence-labeled molecules within a "luminescence marker spot" by means of a mixture with similar, unlabeled molecules during immobilization in such a way that the luminescence intensity from the areas of the luminescence marker spots is of a similar order of magnitude as the luminescence intensity of the from the measuring ranges provided for an analyte detection.
  • the density and concentration of the luminescence-labeled molecules within the "luminescence marker spots" within an array should preferably be uniform across the entire sensor platform.
  • its spatial resolution is essentially determined by the density of the "luminescence marker spots" within an array or on the entire sensor platform.
  • the distance and / or the size of different "luminescence marker spots” are preferably matched to the desired spatial resolution when determining the luminescence intensities from the discrete measurement areas.
  • each array on the sensor platform include at least one "luminescent marker spot”. It is advantageous if there is at least one adjacent “luminescence marker spot” for each segment of measurement areas for determining an analyte.
  • the geometric arrangement of the “luminescence marker spots” within an array or on the sensor platform is that each array comprises a continuous row and / or column of “luminescence marker spots” parallel and / or perpendicular to the direction of propagation of the coupled excitation light, for determining the two-dimensional distribution of the coupled excitation light in the area of said array.
  • the precautions for spatially resolved referencing of the excitation light intensity available in the measuring ranges include averaging over several spatially resolved reference signals.
  • kits according to the invention relate to precautions to calibrate luminescence signals recorded in the presence of one or more analytes.
  • said precautions for the calibration of luminescences generated as a result of the binding of one or more analytes or as a result of the specific interaction with one or more analytes in the near field of layer (a) include predetermined number of arrays. For example, it is possible that 8-12 arrays of a sensor platform are provided for calibration purposes.
  • the kit according to the invention enables a further possibility of calibration that has not been described so far. This consists in the fact that it is essentially not necessary to put a large number of calibration solutions with different, known concentrations on one or more arrays, but in the measuring ranges provided for calibration purposes, the biological or biochemical or synthetic recognition elements used for analyte detection in known, but to immobilize different local concentrations.
  • a calibration curve can be generated for recognition elements in a single constant immobilization density, it is in principle possible to add such a standard curve, which reflects the binding activity and frequency of the binding events between an analyte and its detection elements, by adding a single calibration solution to an array with detection elements in a different immobilization density to create. It is essential for the feasibility of this simplified type of calibration that the binding behavior between an analyte and its recognition elements is precisely known, and that the variation, ie the range between the lowest and highest immobilization density in the measuring ranges provided for an analyte is sufficient for calibration Cover the entire work area of an assay intended for analyte detection.
  • the invention therefore furthermore relates to a kit which is characterized in that in one or more arrays there are in each case a plurality of measurement areas with biological or biochemical or synthetic recognition elements immobilized there in a different, controlled density for the detection of an analyte common to these measurement areas. It is particularly preferred that with known concentration dependency of the binding signals between an analyte and its biological or biochemical or synthetic recognition elements and a sufficiently large "variation" of these recognition elements immobilized in different controlled densities in different measurement areas of an array by adding a single calibration solution to this array a calibration curve can be created for this analyte.
  • kits In another embodiment of the kit according to the invention, several measurement areas of different sizes (diameters) are provided for the detection of one or more analytes. Due to the known fact that the signal intensity to be expected (signal height per unit area of the measuring range provided for this) increases with decreasing area of the measuring range, this enables Embodiment an enlargement of the dynamic range for the detection of said analytes.
  • one or more arrays comprise one or more measuring ranges, which serve for the detection of an analyte of known concentration added to a sample for calibration purposes.
  • This embodiment is comparable to the addition of so-called known standards in analytical separation processes.
  • differences in the binding signals of this known additional analyte e.g. as a result of variations in the physical properties of the sample such as viscosity etc.
  • this embodiment is also suitable for a combination with the variant described above.
  • Another object of the invention is an analytical system with any embodiment of the kit according to the invention, characterized in that it additionally comprises at least one detector for detecting one or more luminescence from the grating waveguide structure.
  • the invention in particular relates to an analytical system for determining one or more luminescences
  • At least one detector for detecting the light emanating from one or more measuring ranges (d) on the sensor platform.
  • a possible embodiment of the analytical system is characterized in that that the excitation light is radiated to the measurement areas in an incident light or transmission light arrangement.
  • the detection of the luminescent light is carried out in such a way that the luminescent light coupled out from a grating structure (c) or (c ') is also detected by the detector.
  • a preferred embodiment of the analytical system according to the invention is characterized in that the excitation light emitted by the at least one excitation light source is essentially parallel and at the resonance angle for coupling into the optically transparent layer (a) onto a grating structure (c) modulated in layer (a) ) is irradiated.
  • the excitation light is expanded by at least one light source with an expansion optics to form an essentially parallel beam and, at the resonance angle, for coupling into the optically transparent layer (a) to a large-area grating structure (c) modulated in layer (a) ) is irradiated.
  • Another possible embodiment is characterized in that the excitation light from the at least one light source through one or, in the case of several light sources, optionally a plurality of diffractive optical elements, preferably Dammann gratings, or refractive optical elements, preferably microlens arrays, into a multiplicity of Individual beams of the same intensity as possible of the partial beams originating from a common light source are broken down, each of which is radiated essentially parallel to one another onto grating structures (c) at the resonance angle for coupling into layer (a).
  • a plurality of diffractive optical elements preferably Dammann gratings, or refractive optical elements, preferably microlens arrays
  • a further development is characterized in that two or more light sources with the same or different emission wavelength are used as excitation light sources. It is preferred that at least one spatially resolving detector is used for the detection, for example from the group formed by CCD cameras, CCD chips, photodiode arrays, avalanche diode arrays, multichannel plates and multichannel photomultipliers.
  • the invention comprises analytical systems which are characterized in that optical components from the group are used between the one or more excitation light sources and the sensor platform as a base plate, as part of a kit according to the invention, and / or between said base plate and the one or more detectors.
  • those of lenses or lens systems for shaping the transmitted light bundles planar or curved mirrors for deflecting and, if necessary, additionally for shaping light bundles, prisms for deflecting and possibly for spectrally splitting light bundles, dichroic mirrors for spectrally selective deflection of parts of light bundles, neutral filters for regulation the transmitted light intensity, optical filters or monochromators for spectrally selective transmission of parts of light beams or polarization-selective elements for the selection of discrete polarization directions of the excitation - Or luminescent light are formed.
  • the light can be excited continuously. However, it is preferred that the excitation light is irradiated in pulses with a duration of between 1 fsec and 10 minutes.
  • a further development of the analytical system is characterized in that the emission light from the measuring ranges is measured in a temporally resolved manner.
  • irradiation and detection of the emission light from all measuring ranges take place simultaneously.
  • Another embodiment is characterized in that the irradiation of the Excitation light on and detection of the emission light from one or more measurement areas is carried out sequentially for single or multiple sample containers. It is also possible for irradiation of the excitation light and detection of the emission light from one or more measurement areas to take place several times sequentially within a single sample container.
  • sequential excitation and detection takes place using movable optical components, which is formed from the group of mirrors, deflection prisms and dichroic mirrors.
  • Sequential excitation and detection can also be carried out using movable glass fibers or glass fiber bundles with which the excitation or luminescent light is sequentially fed to or removed from one or more measuring ranges.
  • a spatially resolving detector is not absolutely necessary, but in this case a simple detector such as a conventional photomultiplier or a photodiode or an avalanche photodiode can be used.
  • sequential excitation and detection is carried out using a scanner that is essentially true to the angle and focus.
  • Another embodiment of an analytical system with sequential excitation and detection is characterized in that the arrangement according to the invention is moved between steps of sequential excitation and detection.
  • the analytical system according to the invention additionally comprises feed means in order to bring the one or more samples into contact with the measurement areas on the sensor platform.
  • feed means in order to bring the one or more samples into contact with the measurement areas on the sensor platform.
  • a further development of the analytical system is characterized in that containers are provided for reagents which are wetted during the method for the detection of the one or more analytes and brought into contact with the measurement areas.
  • containers are provided for reagents which are wetted during the method for the detection of the one or more analytes and brought into contact with the measurement areas.
  • a special embodiment consists in that these additional containers for said reagents are arranged in the body to be brought together with the sensor platform as the base plate.
  • Another object of the invention is a method for the simultaneous qualitative and / or quantitative detection of a large number of analytes with a kit according to the invention in accordance with one of the described embodiments and / or using an analytical system according to the invention, characterized in that one or more of said analytes investigating liquid samples are brought into contact with the measurement areas on a sensor platform as part of said kits, the excitation light intensity available in said measurement areas is referenced in a spatially resolved manner and, if appropriate, one or more luminescences generated in the near field of layer (a) from those with said sample or said Samples brought into contact with measurement areas as a result of the binding of one or more analytes to the biological or biochemical or synthetic recognition elements immobilized in said measurement areas or the interaction between them n said analytes and said immobilized recognition elements are calibrated.
  • the excitation light is coupled to the measurement areas via the grating structure (c) in the optically transparent layer (a).
  • a possible embodiment of the method according to the invention is characterized in that the sensor platform has uniform, unmodulated areas of the Layer (a) comprises, which are preferably arranged in the direction of propagation of the excitation light coupled in via a grating structure (c) and guided in layer (a).
  • (1) the isotropically emitted luminescence or (2) the luminescence or luminescence of both components (1) and (2) coupled into the optically transparent layer (a) and coupled out via grating structures (c) are measured simultaneously.
  • a luminescent dye or luminescent nanoparticle is used as luminescent label to generate the luminescence, which can be excited and emitted at a wavelength between 300 nm and 1100 nm.
  • the luminescence label be bound to the analyte or in a competitive assay to an analog of the analyte or in a multi-stage assay to one of the binding partners of the immobilized biological or biochemical or synthetic recognition elements or to the biological or biochemical or synthetic recognition elements.
  • Another embodiment of the method is characterized in that a second or even further luminescence label with the same or different excitation wavelength as the first luminescence label and the same or different emission wavelength is used.
  • the second or even more luminescent label can be excited at the same wavelength as the first luminescent dye, but emit at other wavelengths.
  • the excitation spectra and emission spectra of the luminescent dyes used overlap only slightly or not at all.
  • a variant of the method consists in that charge or optical energy transfer from a first luminescent dye serving as donor to a second luminescent dye serving as acceptor is used to detect the analyte.
  • Another possible embodiment of the method is that the extent of the quenching of one or more luminescences is determined.
  • a further embodiment of the method is characterized in that in addition to the determination of one or more luminescences, changes in the effective refractive index on the measurement areas are determined.
  • a further development of the method is characterized in that the one or more luminescences and / or determinations of light signals are carried out polarization-selectively at the excitation wavelength.
  • the one or more luminescences are measured with a different polarization than that of the excitation light.
  • a preferred embodiment of the method according to the invention is characterized in that the density of the detection elements immobilized in discrete measurement areas for the detection of different analytes on different measurement areas is selected such that the luminescence signals when detecting different analytes in a common array are of the same order of magnitude, that is to say that the The associated calibration curves for the analyte determinations to be carried out simultaneously can be recorded without changing the optoelectronic system settings.
  • arrays of measurement areas are divided into segments of one or more measurement areas for the determination of analytes and measurement areas for referencing, ie determination of physical parameters and / or chemical differences between different applied samples.
  • one or more arrays can comprise segments of two or more measurement areas with biological or biochemical or synthetic recognition elements of the same type within the segment for analyte determination or referencing.
  • a segment can also contain several discrete measurement areas with different detection elements.
  • a possible variant of the method according to the invention is that different analytes from a common group, such as different cytokines by their binding to different immobilized anti-cytokine antibodies, are determined simultaneously on one or more segments of an array or one or more arrays.
  • Such an embodiment of the method according to the invention is advantageous for such applications, in which one or more measurement areas of a segment or an array are assigned to the determination of the same analyte and whose immobilized biological or biochemical recognition elements have different affinities for said analyte.
  • the recognition elements are expediently selected such that their affinities for different, (bio) chemically similar analytes change in different, characteristic ways.
  • the identity of the analyte can then be determined from the entirety of the signals from different measurement areas with different detection elements for a single analyte, in a manner comparable to a fingerprint.
  • Another possible variant is characterized in that on one or more segments of an array or one or more arrays different analytes from different groups, such as pharmaceutical preparations ("drugs") for the treatment of a disease and / or its metabolites and / or the detection substances for this disease, such as so-called “marker proteins”, are determined.
  • two or more identical measuring ranges are provided within a segment or array for the detection of each analyte or for physical or chemical referencing.
  • Said identical measuring ranges can be arranged, for example, in a continuous row or column or diagonals of an array or segment of measuring ranges.
  • said identical measurement ranges are arranged statistically within an array or segment of measurement ranges.
  • a possible embodiment of the method according to the invention is characterized in that the spatially resolved referencing of the excitation light intensity available in the measurement areas comprises the simultaneous or sequential generation of an image of the light emitted by the sensor platform at the excitation wavelength. It is preferred that the image of the excitation light emitted by the sensor platform is created via the same optical path as the detection of the luminescence originating from the measurement areas.
  • Excitation light intensity includes the simultaneous or sequential creation of an image of the light emitted by the sensor platform at the luminescence wavelength.
  • a further embodiment is characterized in that the precautions for spatially resolved referencing of the excitation light intensity available in the measurement areas comprise the simultaneous or sequential creation of an image of the light emitted by the sensor platform at an excitation wavelength other than for excitation of a luminescence. It is preferred that the excitation wavelength for the spatially resolved referencing is selected such that luminescent molecules used in the course of the method for the detection of one or more analytes or for the purposes of referencing or calibration have no absorption or only the lowest possible absorption at said wavelength, so that Effects of "photochemical bleaching" can be avoided or minimized.
  • the spatial resolution of the image of the excitation light emitted by the sensor platform on the sensor platform is better than 100 ⁇ m, preferably better than 20 ⁇ m.
  • Another object of the method according to the invention is that the spatially resolved referencing of the excitation light intensity available in the measuring ranges by means of "luminescence marker spots", i.e.
  • the luminescence intensity is determined from measurement areas with pre-immobilized (i.e., already applied in these measurement areas before adding a sample) luminescence-labeled molecules.
  • the “luminescence marker spots” are applied in a grid that spans the entire sensor platform.
  • a further development of the method according to the invention is that the density of the luminescence-labeled molecules is selected by means of a mixture with similar, unlabeled molecules during the immobilization in such a way that the luminescence intensity from the areas of the luminescence marker spots is more similar Order of magnitude such as the luminescence intensity of the measuring ranges provided for analyte detection.
  • a preferred embodiment of the method is characterized in that the density and concentration of the luminescence-labeled molecules within the "luminescence marker spots" are uniform within an array, preferably on the entire sensor platform.
  • a known fact is that a luminescent molecule can experience only a limited number of cycles of excitation by an external excitation light and its subsequent deactivation, in the form of the emitted luminescence, before it is photochemically destroyed, i.e. is converted into another, generally no longer luminescent, molecule.
  • This process is commonly referred to as "photobleaching".
  • the number of possible activation and deactivation cycles is an average size characteristic of a certain type of molecule (similar to the half-life of a radioactive substance).
  • the spatially resolved referencing of the excitation light intensity available in the measurement areas comprises averaging over a plurality of spatially resolved reference signals.
  • the one or more samples and the detection reagents to be used in the detection method can be added sequentially in several steps. It is preferred that the one or more samples are preincubated with a mixture of the various detection reagents for determining the analytes to be detected in said samples and these mixtures then in one single addition step to the arrays provided on the sensor platform.
  • a preferred embodiment of the method according to the invention is characterized in that the concentration of the detection reagents, such as, for example, secondary detection antibodies and / or luminescence labels and optionally additional luminescence-labeled detection reagents in a sandwich immunoassay, is selected such that the luminescence signals upon detection of different analytes in a common one Arrays are of the same order of magnitude, which means that the associated calibration curves for the analyte determinations to be carried out simultaneously can be recorded without changing the optoelectronic system settings.
  • the concentration of the detection reagents such as, for example, secondary detection antibodies and / or luminescence labels and optionally additional luminescence-labeled detection reagents in a sandwich immunoassay
  • Another object of an embodiment of the method according to the invention is that the calibration of luminescences generated as a result of the binding of one or more analytes or as a result of the specific interaction with one or more analytes in the near field of layer (a) involves the addition of one or more calibration solutions with known concentrations Analytes to be determined on the same or different measuring areas or segments of measuring areas or arrays of measuring areas on a sensor platform, to which the one or more samples to be examined are supplied in the same or a separate addition step.
  • a particular embodiment of the method is characterized in that the calibration of luminescences generated as a result of the binding of one or more analytes or as a result of the specific interaction with one or more analytes in the near field of layer (a), the addition of an optionally additional analyte of known concentration to a concentration or several samples to be examined for detection on one or more measuring areas of the sensor platform designated for this purpose.
  • Another preferred embodiment of the method is characterized in that the calibration of luminescence generated as a result of the binding of one or more analytes or as a result of the specific interaction with one or more analytes in the near field of layer (a) compares the luminescence intensities after adding an unknown and a Control sample, such as a "wild type" DNA sample and a "mutant DNA” sample. It is possible that the unknown sample and the control sample are added to different arrays.
  • Another variant of this method is characterized in that the unknown sample and the control sample are added sequentially to the same array.
  • a regeneration step is generally necessary between the addition of the unknown sample and the control sample, i.e. the dissociation of recognition element-analyte complexes formed after the addition of the first sample, followed by the removal of the dissociated analyte molecules from the sample containers before the addition of the second sample can take place.
  • several samples on an array of measurement areas can be examined for their analytes in sequential form.
  • Another possible embodiment of the method is that the unknown sample and the control sample are mixed and then the mixture is fed to one or more arrays of a sensor platform.
  • a further development of the method according to the invention is characterized in that the analytes to be detected in the unknown and the control sample are detected by means of luminescence labels of different excitation and / or luminescence wavelength for the unknown and the control sample.
  • the detection is carried out using two or more luminescence labels with different excitation and / or luminescence wavelengths.
  • the use of several different luminescence labels can also be advantageous when determining different analytes from a common group.
  • a further preferred embodiment of the method according to the invention therefore consists in that, for example to determine the cross-reactivity between different analytes from a common group, such as, for example, the cytokines, and their recognition elements, such as, for example, anti-cytokine antibodies, the detection using two or more luminescence labels with different excitation and / or luminescence wavelengths.
  • the kit according to the invention with the large number of measuring ranges on a sensor platform opens up the possibility of a simplified form of calibration for the qualitative and / or quantitative determination of one or more analytes on one or more arrays.
  • this new form of calibration of the signals from a sensor platform according to the invention the addition of only a single calibration solution is required.
  • a further, preferred variant of the method according to the invention is characterized in that the cooling is due to the binding of one or more Analytes or luminescence generated as a result of the specific interaction with one or more analytes in the near field of layer (a) comprises determining the luminescence intensity due to the presence of one or more analytes present in a series of samples in a substantially constant concentration.
  • so-called "housekeeping genes” are known in DNA analysis, in particular for comparing so-called “wild type” and “mutant” samples, the frequency of which is essentially constant in a series of samples of similar origin (tissue, type of organism, etc.) is.
  • immunoanalytics are certain immunoglobulins, the concentration of which changes only slightly between different samples of a common type of organism.
  • Part of the invention is a method according to one of the aforementioned embodiments for the simultaneous or sequential, quantitative or qualitative determination of one or more analytes from the group of antibodies or antigens, receptors or ligands, chelators or "histidine tag components", oligonucleotides, DNA or RNA strands, DNA or RNA analogs, enzymes, enzyme cofactors or inhibitors, lectins and carbohydrates.
  • Possible embodiments of the method are also characterized in that the samples to be examined naturally occurring body fluids such as blood, serum, plasma, lymph or urine or egg yolk or optically cloudy liquids or tissue fluids or surface water or soil or plant extracts or bio or synthesis process broths or from biological Tissue parts or taken from cell cultures or extracts.
  • body fluids such as blood, serum, plasma, lymph or urine or egg yolk or optically cloudy liquids or tissue fluids or surface water or soil or plant extracts or bio or synthesis process broths or from biological Tissue parts or taken from cell cultures or extracts.
  • the invention furthermore relates to the use of a kit according to the invention and / or an analytical system according to the invention and / or a method according to the invention for quantitative or qualitative analyzes for determining chemical, biochemical or biological analytes in screening methods in pharmaceutical research, combinatorial chemistry, clinical and preclinical Development, real-time engagement studies and Determination of kinetic parameters in affinity screening and in research, for qualitative and quantitative analyte determinations, in particular for DNA and RNA analysis, for the preparation of toxicity studies and for the determination of gene or protein expression profiles as well as for the detection of antibodies, antigens, Pathogens or bacteria in pharmaceutical product development and research, human and veterinary diagnostics, agrochemical product development and research, symptomatic and presymptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for therapeutic drug selection, for the detection of pathogens, pollutants and Pathogens, especially Salmonella, prions, viruses and bacteria, in food and environmental analysis.
  • the main component of a kit according to the invention is a rectangular sensor platform with the outer dimensions 113.5 mm x 75.0 mm x 0.7 mm thickness, connected with an 11 mm thick polycarbonate (PC) layer, which is colored black to suppress scattered light artifacts.
  • PC polycarbonate
  • the recesses are arranged in the form of 12 columns and 8 rows on one level, so that the combination of the sensor platform and the PC structure comprises a total of 96 sample containers.
  • Continuous surface relief gratings were created in the substrate at intervals of 9 mm, with a width of 0.5 mm (in the direction of propagation of the excitation light to be coupled into the layer (a) of the sensor platform via the lattice structure). These gratings have a period of 360 nm and a depth of 12 nm, with the orientation of the grating lines parallel to the columns of the wells.
  • the wave-guiding, optically transparent layer (a) made of Ta 2 O 5 on the optically transparent layer (b) has a refractive index of 2.11 at 633 nm (layer thickness 150 nm).
  • the lattice structure of the optically transparent layer (b) is transferred almost to scale on a 1: 1 basis into the surface of the applied layer (a).
  • the surface of the sensor platform is wet-chemically cleaned before being assembled with the polycarbonate structure, first several times with isopropanol, then with concentrated sulfuric acid, which contains 2.5% ammonium peroxodisulfate.
  • a monomolecular layer (monolayer) of mono-octadecyl phosphate is then applied as an adhesion-promoting layer in a self-assembly process (self-assembly) to the hydrophilic waveguide surface.
  • This surface modification leads to a hydrophobic surface (contact angle approx. 110 ° with water).
  • the process of surface modification has been described in more detail in the literature (D. Brovelli et al., Langmuir 15 (1999) 4324-4327).
  • the recognition elements for the detection of various human interleukins are in a concentration between 300 and 1000 ⁇ g / ml reconstituted in ten percent phosphate buffered saline (PBS, pH 7.4).
  • the antibody solutions are then diluted to varying degrees in 10% PBS (pH 7.4), which is determined by the affinity of the respective antibody for the corresponding antigen.
  • the required concentrations (100 ⁇ g / ml for anti-hIL-2 and anti-hIL-6 or 50 ⁇ g / ml for anti hIL-4 antibodies) were previously determined in single interleukin immunoassays. This is intended to ensure that the dynamic range of the signal intensities to be expected in an assay for the simultaneous detection of all three interleukins is of the same order of magnitude within an array.
  • This aspect of the example demonstrates that by suitable selection of the immobilization density of different detection elements in discrete measurement areas, with different affinities for the analytes to be detected, it is possible that the dynamic range of the signal intensities to be expected in an assay for the simultaneous detection of a large number of different ones Analytes within an array is of the same order of magnitude.
  • the mixture is incubated for 15 min in a saturated steam atmosphere, then the non-protein-covered, hydrophobic surface of the sensor platform with a solution of bovine serum albumin (BSA) in PBS (1 mg / ml, pH 7.4) with the addition of 0.05 % Tween 20, saturated, to minimize non-specific binding of detection antibodies in the later detection method, then washed with H 2 O and dried with nitrogen.
  • BSA bovine serum albumin
  • the diameter of the spots is approx. 220 ⁇ m.
  • a single array comprises three different types of recognition elements (for the recognition of hIL-2, hIL-4 and hIL-6) as well as "luminescence marker spots" with Cy5 fluorescence-labeled bovine serum albumin (Cy5-BSA).
  • Cy5-BSA Cy5 fluorescence-labeled bovine serum albumin
  • the immobilization density of the Cy5-BSA is chosen so that the ruorescence intensity of these “luminescence marker spots” is also in the dynamic range of the expected signal intensity changes of the interleukin assay.
  • a 25 picomolar solution of Cy5-BSA is determined as an optimal concentration of the Cy5-BSA in the immobilization solution, with a labeling rate of 10 Cy5 molecules per BSA molecule. Furthermore, it is found that to achieve a homogeneous distribution of the fluorescence-labeled BSA molecules in the "luminescence marker spots", the use of a mixture of unlabeled and fluorescence-labeled B SA molecules for the immobilization solution is much more suitable than the use of a solution with only the fluorescence-labeled one Protein, with a correspondingly lower protein concentration.
  • the "luminescence marker spots” are arranged in four rows, each with four spots, parallel to the rows of the detection element spots.
  • the "luminescence marker spots” serve for referencing the excitation light available in the adjacent measurement areas for analyte detection; their arrangement in rows parallel to the direction of propagation of the excitation light to be coupled into the layer (a) and to be guided there also serves to determine the attenuation (attenuation) of the excitation light in the direction of propagation.
  • two columns of "luminescence marker spots”, each with seven replicas, are arranged at the beginning and at the end of the array, in the direction of propagation of the coupled and guided excitation light. They serve to determine the homogeneity of the available excitation light intensity parallel to the lines of the coupling-in grating.
  • Example 2 Analytical system with a kit according to the invention
  • the sensor platform is mounted on a computer-controlled adjustment unit, which allows translation parallel and perpendicular to the grid lines and rotation about an axis of rotation parallel to the grid lines of the sensor platform.
  • a computer-controlled adjustment unit which allows translation parallel and perpendicular to the grid lines and rotation about an axis of rotation parallel to the grid lines of the sensor platform.
  • a shutter in the light path to block the light path if no measurement data are to be recorded.
  • neutral filters or polarizers can be placed in the light path at this point or at other positions in the further path of the excitation light to the sensor platform in order to vary the excitation intensity step-wise or continuously.
  • the excitation light beam of a helium-neon laser at 632.8 nm is widened in one dimension with a cylindrical lens and passed through a slit-shaped diaphragm (0.5 mm x 7 mm opening) so as to Generate light beams of approximately rectangular cross-section and approximately homogeneous cross-sectional intensity.
  • the polarization of the laser light is aligned parallel to the grid lines of the sensor platform to excite the TE 0 mode under coupling conditions.
  • the excitation light is directed from the back of the sensor platform, i.e.
  • the angle between the sensor platform and the irradiated excitation light bundle is adjusted for maximum coupling into the optically transparent layer (a) by rotation about the aforementioned axis of rotation. With the aforementioned parameters of the sensor platform, the resonance angle for the coupling in air is approximately 2.6 °.
  • a CCD camera (Ultra Pix 0401E, Astrocam, Cambridge, UK) with Peltier cooling (operating temperature -30 ° C), with a Kodak CAF chip KAF 0401 El serves as the spatially resolving detector.
  • the format of a sandwich assay is selected for the specific detection of the interleukins to be detected.
  • the calibration solutions, as well as the samples with unknown, to be determined concentrations of the three interleukins as analytes, are then mixed with 50 ⁇ l of a solution containing a mixture of three secondary polyclonal detection antibodies (5 x 10 "10 M biotinylated anti-hIL- 2 antibodies, 10 "10 M biotinylated anti-hIL-4 antibody and 10 " 10 M biotinylated anti-hIL-6 antibody in PBS (pH 7.4), with 0.1% BSA and 0.05% Tween 20).
  • This aspect of the example demonstrates that by appropriately selecting the concentrations of the detection reagents, it is possible for all to be in one The analyte to be assayed at the same time, the expected ruorescence intensities, due to their specific binding to the respective detection elements immobilized in discrete measurement areas, are of the same order of magnitude, ie that the corresponding calibration curves can be recorded without changing the optoelectronic system settings.
  • the mixed solutions prepared are then incubated at room temperature in the dark for one hour before the incubates (100 ⁇ l each) are filled into the sample containers.
  • the calibration solutions are filled in increasing concentration into the sample containers for the arrays AI to Hl (microtiter plate nomenclature, see Figure 1) of the sensor platform, whereby the 88 samples to be investigated are distributed with unknown concentrations of the three interleukins as analytes in the remaining sample containers A2 to H12 , After another two hours of incubation at room temperature in the dark, the arrays are read out.
  • the sensor platform with the sample containers generated thereon and the solutions therein are mounted on the adjustment unit described above within the analytical system.
  • the sensor platform is adjusted to the maximum coupling of the excitation light via the grating structure assigned to the respective array, which is checked with the position of the filter changer for the excitation wavelength.
  • the intensity of the fluorescent light is then measured from the measurement areas (spots) of the arrays with the position of the filter changer for the luminescence wavelength.
  • the arrays in the other sample containers are read out sequentially, by translating the sensor platform to the next position for reading out the luminescence signals from the next sample container. Evaluation and referencing:
  • the image analysis is carried out using commercially available image processing software
  • Array each have four integral ruorescence intensity values, from which to statistical
  • the two Cy5-B SA reference spots (“luminescence marker spots”) from the first column of the array before and the last column after the respective row, each with four measuring ranges for determining the component, are evaluated and averaged. This averaged reference value is used in each case for the correction of the luminescence signals from the measurement ranges in the same row for the analyte determination.
  • the mean values of the Cy5-B SA reference spots are formed for each array before and after the interleukin measurement areas in the same row in each case.
  • An average is again formed from each of these 96 averaged reference values for each of the three analytes.
  • the individual correction factor for the measured values for analyte determination in an array then results as a quotient from the local reference value and the last-mentioned mean value. Multiplying by this correction factor compensates for the local differences in the available excitation light intensity on a common sensor platform.
  • the luminescence intensities corrected by the method described above are normalized to the value 1 at an interleukin concentration of 0 pg / ml.
  • Figure 2 shows an example of the uncorrected raw data obtained for the detection of interleukin 4 for the calibration of this multianalyt immunoassay, in which the integral ruorescence intensity values as a function of the hIL-4 concentration are plotted.
  • Figure 3 shows the corrected calibration data generated using the averaging described.
  • Figure 4 shows the continuous curve adjusted to this corrected data as a solid curve.
  • the empty symbols show the calibration signals determined with three different sensor platforms after correction.
  • the filled circles each represent the mean values formed therefrom, at the different hIL-4 concentrations.

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Abstract

L'invention concerne différents formes de réalisation d'un kit servant à la détection simultanée, qualitative et/ou quantitative, d'une pluralité d'analytes. Ce kit comprend une plate forme de détecteur qui présente un guide d'ondes optique à couches minces comportant au moins une couche (a) qui est transparente à au moins une longueur d'ondes d'excitation, située sur une couche (b) également transparente à au moins cette longueur d'ondes d'excitation, possédant un indice de réfraction inférieur à celui de la couche (a), et au moins une structure réticulaire (c) modulée dans la couche (a), servant à injecter la lumière d'excitation dans la couche (a). Ce kit comprend également un ensemble d'éléments de reconnaissance biologiques ou biochimiques ou synthétiques, immobilisés sur la couche (a), dans des zones de mesure discrètes (d) directement ou non sur une couche favorisant l'adhérence. Ces éléments servent à la reconnaissance et/ou à la liaison spécifiques desdits analytes et/ou à l'interaction spécifique avec lesdites analytes. Ce kit comprend en outre des moyens pour le référencement à résolution spatiale de l'intensité de la lumière d'excitation disponible dans les zones de mesure, ainsi qu'éventuellement des moyens pour le calibrage d'une ou plusieurs luminescences produites à la suite de la liaison d'un ou plusieurs analytes ou à la suite de l'interaction spécifique avec un ou plusieurs analytes dans le champ proche de la couche (a). Un échantillon liquide, dans lequel lesdits analytes doivent être détectés, est mis en contact, avec lesdites zones de mesure sur la plate-forme de détecteur, directement ou après avoir été mélangés avec d'autres réactifs. L'invention concerne en outre des systèmes analytiques faisant appel à ce kit, ainsi que des procédés mis en oeuvre avec ce kit pour détecter un ou plusieurs analytes, et l'utilisation de ces systèmes.
EP01940527A 2000-06-02 2001-05-25 Kit et procede pour la detection d'une pluralite d'analytes Withdrawn EP1287360A2 (fr)

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US20080212070A1 (en) 2008-09-04
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JP2004510130A (ja) 2004-04-02
JP4812223B2 (ja) 2011-11-09
US20030148542A1 (en) 2003-08-07
WO2001092870A3 (fr) 2002-10-03
US7645612B2 (en) 2010-01-12

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