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
  • G01N21/648—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
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/80—Fluorescent dyes, e.g. rhodamine
• • 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
  • Y10S436/815—Test for named compound or class of compounds
• • 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
  • Y10S436/815—Test for named compound or class of compounds
  • Y10S436/817—Steroids or hormones

Definitions

• the present invention relates to a method for determining substances based on the evanescent field method and a cuvette, a microtiter plate, a solution and a kit for use in the method according to the invention.
• the present invention can be used in particular in diagnostics and analysis.
• ELISA enzyme-linked immunoabsorbent assay
• a second reaction partner for example another antibody for the substance to be investigated is then brought into contact with the carrier, this reaction partner being marked with an enzyme which permits colorimetric detection.
• the reaction of this second reaction partner with the substance to be examined which is coupled to the surface of the carrier produces a colored product which can be optically evaluated.
• Standardized plastic plates often made of polystyrene, with 96 wells are usually used as the solid phase. The surface of the plastic wells binds proteins in the nanogram range through adsorption, which is a sufficient amount for immunological detection.
• ELISAs show very good results in terms of sensitivity and specificity, the detection limits that can be achieved are in the nanogram range or below. There are various embodiments of assays based on this principle. Depending on the question, antigens or antibodies are detected.
• a major disadvantage of the ELISA is the handling of the test, since different reagents have to be added to the wells in succession and then removed again. A total of ten or more pipetting, washing and incubation steps may be required. That is why ELISAs are time-consuming and labor-intensive and must be carried out with great care by specially trained personnel.
• Another disadvantage of the ELISA is the time required for the sum of the incubation and washing steps for an assay or test, which normally takes one to several hours.
• the interaction of, for example, biomolecules on a surface can be observed directly using the evanescence field method.
• the interaction of the reactant in solution with a solid matrix surface is measured.
• the binding of the ligand can be measured physically as a surface plasmon resonance ("surface plasmon resonance") without a time delay (in "real time”).
• surface plasmon resonance a surface plasmon resonance
• the advantages compared to an ELISA are the omission of further pipetting steps after the addition of the reagents and the elimination of the waiting steps. So far, however, complex devices and multilayer sensor chips with special surface chemistry have been required for such measurements. These disadvantages have prevented the method from being used in routine diagnostics.
• the present invention is therefore based on the object of a method for
• a method for determining substances comprising the steps - providing a surface which comprises at least one reaction partner R 1 for a reaction partner R 2 bound to the surface, contacting the surface with a solution which contains at least the substance to be determined, at least a fluorophore-containing compound and at least one dye which absorbs in the absorption and / or emission region of the fluorophore, in which a complex is formed on the surface of the reaction partner R 1 by means of the reaction partner R 2 and in which this complex in addition to the reaction partner R 1 comprises at least the substance to be determined and the at least one fluorophore-containing compound, and - excitation of the fluorophore bound on the surface by the evanescent field of a light source and measurement of the fluorescence generated.
• Figure 1 is a schematic representation of an embodiment of the cuvette according to the invention and the inventive method according to one embodiment.
• Figure 2 shows a schematic representation of a further embodiment of the method according to the invention.
• FIG. 3 shows the intensity curve of electromagnetic radiation within a dye solution in accordance with Lambert-Beer law.
• FIG. 4 shows a double logarithmic representation of the intensity curve as a function of the penetration depth of an evanescent wave and a wave weakened by absorption in accordance with Lambert-Beer law.
• Figure 5 shows the absorption spectra of a number of dyes.
• FIG. 6 shows the determination of the optimal concentration of a dye for use according to the method according to the invention.
• FIG. 7 shows a reaction kinetics of the attachment of a protein to reaction partners R 1 bound on the surface, measured by means of the method according to the invention.
• FIG. 8 shows a comparison measurement for the reaction kinetics according to FIG. 7, which was measured as in FIG. 7. However, in this comparative example, the surface was not coated with a reactant R 1 for the protein.
• a surface which comprises at least one reaction partner R 1 bound or immobilized.
• Bound means preferably that the reactant R 1 adheres to the surface by adsorption (direct adsorption).
• the reaction partner R 1 can, however, also be bound to the surface via a bridge member, for example a protein, such as an antibody or an antigen.
• the reactant R 1 can also be bound to the surface by a covalent bond. In the case of an acrylate surface, for example, this can be brought about by reaction with a carbodiimide.
• bound means the adherence of a reaction partner or a compound to a surface or to a further reaction partner and / or compound and includes both covalent and non-covalent interactions, such as interactions due to ionic, polar or non-polar interactions ,
• the reactant R 1 can be applied to the surface by conventional methods.
• a protein serving as reaction partner R 1 can be coated on the surface (coating).
• the reaction partner R 1 can preferably be bound to the surface by adsorption or by covalent binding.
• the surface is preferably treated with a further solution, by means of which parts of the surface not afflicted with the reactant R are blocked or blocked, for example by a further protein which essentially does not contain the solution to be contacted Components responded.
• the protruding surface is, for example, an inside of a concave container, such as a cuvette or a well (well) of a microtiter plate.
• the reaction partner R 1 bound to the surface can form a complex on the surface by means of a reaction partner R 2 , this complex comprising at least the substance to be determined and the at least one fluorophore-containing compound in addition to the reaction partner R 1 .
• the complex with the substance to be determined is "anchored", ie fixed, by the reaction partner R 1 bound to the surface, and can at the same time be detected by labeling with the fluorophore-containing compound.
• a “complex” or “conjugate” is understood to mean a molecular linkage or binding together of two or more preferably chemical or biochemical substances. Training the
• Reactions particularly preferably by antigen-antibody reactions.
• the term “implementation” encompasses both covalent and non-covalent interactions of two or more reaction partners, it being possible for both types of interaction to coexist within a complex or conjugate.
• non-covalent interaction encompasses both covalent and non-covalent interactions of two or more reaction partners, it being possible for both types of interaction to coexist within a complex or conjugate.
• Van-der-Waals interaction Van-der-Waals interaction, polar and / or ionic interaction of the
• Reaction partners mean.
• reaction partner means a connection with an affinity for another substance.
• the complex in addition to the reaction partner R 1, the complex comprises at least the substance to be determined and the at least one fluorophore-containing compound.
• Reaction partner R 2 has the following options, among others:
• the substance to be determined is the reaction partner R 2 .
• the substance to be determined comprises the reaction partner R 2 , ie the reaction partner R 2 is a partial structure of the substance to be determined.
• the substance to be determined has an affinity or binding site for the reaction partner R 2 .
• Case (2) can thus arise after the binding of the reaction partner R 2 to the substance to be determined.
• a further compound comprises the reaction partner R 2 or has an affinity for the reaction partner R 2 , this further compound further comprising at least one binding site for the substance to be determined.
• the further compound, the substance to be determined and the reaction partner R 2 can be added to the solution as a conjugate or complex (all or only some) or the conjugate forms in the solution.
• the substance to be determined can itself have an affinity for the reaction partner R 1 on the surface and can therefore form a bond directly with this reaction partner R 1 .
• the substance to be determined can bind as reaction partner R 2 to the reaction partner R 1 on the surface. If, for example, the substance to be determined is an antibody, an antigen specific for this antibody can be applied to the surface, or vice versa.
• FIG. 1 shows a schematic representation of an embodiment of the cuvette according to the invention, and the inventive method according to the above embodiment.
• the cuvette 1 has a recess 2, the surface of which comprises 3 reactants R 1 4 bound for the protein to be determined.
• the depression 2 also increases with the surface 3 contacting solution 5, which according to this embodiment comprises a dye 6 and the substance 7 to be determined, which is already present as a conjugate with the fluorophore-containing compound.
• the substance to be determined reacts with the reactant R 1 4 bound on the surface to form a complex 9 on the surface 3.
• a light beam 10 is projected onto the underside of the surface 3, which is totally reflected at the phase interface 11.
• an evanescence field 13 is formed above the surface 3, in which there is essentially only fluorophore bound to the surface in the complex 9.
• the evanescence field does not usually extend over the entire width of the cuvette bottom.
• the evanescence field can have an extent of approximately 1 mm 2 . Due to the excitation of the fluorophores by the evanescence field 13, the fluorophores bound on the surface emit photons 14, which can be amplified and measured, for example, by means of a photomultiplier 15. The fluorescence of the volume 16 is essentially suppressed by the presence of the dye 6.
• the substance to be determined itself has (essentially) no or only slight affinity for the reaction partner R 1 on the surface.
• the solution to be brought into contact with the surface contains a further compound which comprises a reaction partner R 2 and a binding site to the substance to be determined.
• the reaction partner R 2 can bind to the reaction partner R 1 on the surface and thus fixes the substance to be determined indirectly on the surface.
• This further connection thus serves as a bridge between the substance to be determined and the reaction partner R 1 on the surface.
• avidin may be present as the reaction partner R 1 on the surface.
• the further compound then comprises, in addition to a binding site for the substance to be determined, for example biotin, which can bind to the avidin bound on the surface.
• This embodiment has the advantage, for example, that, in contrast to some antibodies and antigens, a surface coated with avidin can be lyophilized and dried or lyophilized is very stable. It also points out System Avidin / Biotin a very high dissociation constant KD. Furthermore, it is possible in this way to always provide an avidin-coated surface for a number of different determinations and to match only the further compound which is brought into contact with the solution with the surface to the substance to be determined.
• Figure 2 shows schematically this embodiment of the method according to the invention.
• the substance 20 to be determined, a dye 22, a fluorophore-containing compound 24 and a further compound 26 are present side by side in the solution brought into contact with the surface.
• the reactant R 1 28 is bound on the surface.
• the further compound and the fluorophore-containing compound attach to the substance to be determined (conjugate 30), and conjugate 30 binds via the reaction partner R 2 present in the further compound 26 to the reaction partner R 1 present on the surface 28 to the complex 32.
• the complex 32 which contains the fluorophore-containing compound 24, is bound to the surface and can be determined by measuring the fluorescence in the evanescent field 34.
• avidin or streptavidin
• biotin all ligands or ligand-binding systems in which, for example, proteins are selective and / or specific binding sites for one or more ligands, such as histidine, histidine tags, are suitable for this embodiment of the method according to the invention.
• the solution to be contacted with the surface further contains at least one fluorophore-containing compound.
• a fluorophore is understood to mean a fluorescent compound, such as a fluorescent dye. Fluorescent proteins and / or low-molecular fluorescent chemical compounds are preferred.
• phycobiliproteins such as allophycocyanin (APC), Cryptofluor Crimson or Cryptofluor Red can be used as fluorescent proteins.
• APC allophycocyanin
• Cryptofluor Crimson Cryptofluor Red
• Cryptofluor Red can be used as fluorescent proteins.
• low-molecular fluorescent compounds for example
• Cy5 or BODIPY (4,4-Diluor-4-bora-3a, 4a-diaza-s-indazene fluorophores) can be called.
• Fluorescent dyes with an absorption in the range from 600 to 700 nm are preferred.
• a fluorophore precursor compound can be used, from which the fluorophore is released before the measurement process, for example by changing the pH or by splitting off a protective group.
• the term fluorophore also includes phosphorescent compounds. If such a phosphorescent compound is used as the fluorophore, the emitted phosphorescence is determined, which takes place at different times from the excitation. It is thus possible to separate the period of irradiation from the period of measurement.
• this fluorophore-containing compound has a binding site for the substance to be determined.
• the fluorophore can be bound to an antibody.
• This fluorophore-containing antibody can preferably react in an antigen-antibody reaction with the substance to be determined, for example a protein, as the antigen.
• the substance to be determined is itself present as a fluorophore-containing compound.
• competition assays can be carried out, which are characterized in particular by a low detection limit.
• the method is suitable for determining biologically active substances, such as hormones, proteins such as antigens, antibodies or haptens, pharmaceuticals, viruses, bacteria, etc.
• biologically active substances such as hormones, proteins such as antigens, antibodies or haptens, pharmaceuticals, viruses, bacteria, etc.
• the method can also be used to detect environmental toxins, toxins, etc.
• the substances to be determined are particularly preferably detected by immunological reactions.
• a complex of at least the first reaction partner R 1 , the substance to be determined and the fluorophore-containing compound forms on the surface. It is then possible to excite the fluorophore bound to the surface by the evanescent field of a light source and to measure the fluorescence of the fluorophore.
• a light beam is directed onto the underside of the surface at such an angle that total reflection occurs at the phase interface of the cuvette / solution.
• an angle of incidence of at least 60 ° to 90 ° is preferred, so that an evanescent field is formed above the surface at a height of up to 400 nm, preferably 200 nm, particularly preferably 50 to 150 nm.
• the incident light is able to excite suitable fluorophores.
• the emitted fluorescent light is amplified and evaluated, for example, with a photomultiplier.
• Monochromatic light can be used as the light source. It should
• Light of a wavelength can be used, which is preferably not with the
• a laser is particularly preferred as the light source, in particular laser which emits light of a wavelength of
• the 40 emit at least 635 nm.
• the solution to be examined is serum, wavelengths of 600 to 700 nm are preferred, since the inherent fluorescence of serum is around 580 nm.
• the increase in the fluorophore bound to the surface can be measured directly in real time as the reaction progresses. Since the amount of fluorophore bound to the surface is directly proportional to the amount of fluorophore-containing compound originally present, the method according to the invention permits the quantitative determination of reactants in solution in real time without additional washing and / or pipetting steps.
• volume is understood to mean the liquid which is outside the evanescence field and which contains unbound fluorophore-containing compounds.
• polarization of the light beam can be rotated in both plastic and glass cuvettes. This leads in particular to reflections of the excitation light when coupling out. So-called vagabond light is created, which together with volume and surface scattering effects can lead to volume stimulation.
• excitation of the fluorophore located in the volume can be prevented if at least one dye is added to the solution to be contacted with the surface, which dye has an absorption in the absorption and / or emission range of the fluorophore.
• a comparison of the penetration depths of evanescent wave and of stray light shows that the volume excitation can be suppressed by adding a dye.
• the absorption of light is physically described by Lambert-Beer law, the intensity of the light decreasing logarithmically with the distance by absorption:
• FIG. 4 shows a comparison of the penetration depths of the evanescent wave with the penetration depth of the light when absorbed by a dye.
• the double logarithmic representation shows the intensity curve as a function of the penetration depth of an evanescent wave (left) and a wave attenuated by absorption (right).
• the ordinate is in the range from -2 to 0, i.e. from 1/100 to 1 Iog10 intensity.
• the parameters used here correspond to technically realizable values.
• the decisive factor for the effectiveness of suppression is the geometric one Distance between the surface part of the cuvette from which light can reach the detector and the penetration points of the stray light into the volume.
• a distance in the range of one millimeter is sufficient for a weakening of scattered light of two orders of magnitude. This distance can be easily maintained by appropriate cell dimensioning.
• the absorption of the dye added to the volume is matched to the absorption and / or emission range of the fluorophore.
• a single dye or a mixture of dyes can be used.
• the absorption range of the fluorophore will usually correlate with the wavelength of the light source used. It is not necessary for the dye to have an absorption maximum in this spectral range; a shoulder in the absorption spectrum may already be sufficient. If, for example, fluorophores such as APC or Cy5 are used, the dye used can have an absorption between 600 nm and 700 nm, such as, for example, brilliant blue (brilliant blue FCF).
• the concentration of the added dye depends on the absorption coefficient of the respective dye in solution and also depends on the frequency of the incident light.
• the concentration of the dye can be adjusted depending on the dye so that the penetrating light can be essentially absorbed within 1 mm above the surface.
• the volume fluorescence and the fluorescence in the evanescent field i.e. the surface fluorescence, measured in each case at different dye concentrations (cf. FIG. 6a).
• the ratio of surface fluorescence to volume fluorescence is then plotted against the concentration of the dye (cf. FIG. 6b).
• the maximum of curve 6b represents the optimal concentration of the dye.
• the “signal / noise ratio” is understood to mean the ratio of surface fluorescence (“signal”) to volume fluorescence (“noise”).
• “Essentially absorbed” can be one Intensity deletion of 70%, preferably 80%, particularly preferably at least 90%.
• the concentration of brilliant blue FCF is preferably at least 0.001 mM.
• the present invention further relates to a cuvette or a microtiter plate for carrying out the method according to the invention.
• the cuvette preferably comprises glass or a plastic, particularly preferably a plastic such as polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polycycloolefin, polyacrylonitrile, polymethyl methacrylate and / or mixtures or blends of these plastics.
• a plastic such as polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polycycloolefin, polyacrylonitrile, polymethyl methacrylate and / or mixtures or blends of these plastics.
• any plastic is suitable which essentially does not absorb any light in the visible range.
• the plastic can also be colored, for example, slightly bluish in order to filter out an emission caused by scattered light.
• Plastic cuvettes can be obtained inexpensively by injection molding and preferably have a reaction volume of 1 to 400 ⁇ l, particularly preferably 5 to 200 ⁇ l.
• the cuvettes or microtiter plates according to the invention are preferably formed in one piece. It can also prove to be advantageous if the inside and / or the emission surface, i.e. the surface from which the emitted beam emerges from the cuvette is / are polished to a surface roughness of preferably at most 10 nm.
• the small size and the low price make it possible to use the method according to the invention in routine diagnostics and analysis.
• a cuvette or microtiter plate can already be prepared and closed in the special label Trade.
• the prepreparation includes the coating of the surface of the cuvette or microtiter plate with the first reaction partner and, if necessary, the subsequent blocking of the non-coated areas.
• the coated cuvette or microtiter plate is particularly preferably lyophilized or dried.
• the at least one dye and / or the at least one fluorophore-containing compound and / or the further compound may also be lyophilized and / or dried in the closed cuvette or microtiter plate, so that only the substance to be examined is present in the measurement Solution must be added.
• the present invention comprises a solution which comprises at least one fluorophore-containing compound and / or at least one dye which absorbs in the absorption and / or emission range of the fluorophore.
• the solution according to the invention can optionally contain a further compound which has at least one binding site to the substance to be determined and which comprises the reactant R 2 .
• the present invention comprises a kit which comprises a prepared cuvette or microtiter plate and / or solutions of the at least one dye and the at least one fluorophore-containing compound and, if appropriate, the further compound which has at least one binding site to the substance to be determined and which comprises the reactant R 2 .
• the at least one dye and the at least one fluorophore-containing compound can be present together in one solution and in two separate solutions.
• the present invention further relates to the use of the method according to the invention for determining reaction kinetics, preferably immunological reactions, and the use of the method in medical or veterinary diagnostics, food analysis, environmental analysis or analysis of fermentation processes.
• the detection of crop protection agents, such as atrazine, in drinking water, the detection of hormones in veal, the detection of hormones, such as HCG, and the direct or indirect detection of viruses, such as hepatitis S and HIV, can be mentioned as examples of specific applications.
• the influence of the concentration of the added fluorophore bound to a protein is determined.
• only fluorophore bound to the surface is present; unbound fluorophore was washed away. A dye was not added to the solution.
• GAMAPC conjugate of allophycocyanin (APC) and Crosslinked, Goat Anti-Mouse IgG (H + L); Molecular Probes, Leiden, Netherlands
• PBS + T 100 mM PO 4 , pH 7.5; 100 mM NaCI, 0.025v / v Tween20
• PBS + T 100 mM PO 4 , pH 7.5; 100 mM NaCI, 0.025v / v Tween20
• Endpoint reaction with washing the chip and measuring the fluorescence. Only bound fluorophore is present in the measurement.
• the surface of cuvettes was coated by leaving 200 ⁇ l human serum 1: 1000 in PBS + at room temperature (RT) on the surface overnight. Then the surface was washed four times with PBS and treated with 1% BSA (Bovine Serum Albumine) Miles enhanced, PBS +, 300 ⁇ l, for one hour at RT.
• BSA Bovine Serum Albumine
• the cuvette is blocked with 1% BSA Miles enhanced, in PBS + 300 ⁇ l, for one hour at RT.
• the reduction in volume excitation depends on the concentration of the brilliant blue dye in the volume. With brilliant blue FCF, at a concentration of 0.04 mm and above, far more than 95% of the volume fluorescence is extinguished.
• a cuvette prepared as in Example 1a) is contacted with GAMAPC in PBS + T overnight at RT and then washed five times with PBS.
• the negative control M1 has 5,000 counts / s.
• Brilliant blue FCF reduces the volume emission from APC from 230,000 counts / s M1 to 5,000 counts / s.
• the specific surface-bound fluorescence is reduced by about 50%.
• the signal-to-noise ratio is significantly improved when adding brilliant blue FCF.
• reaction kinetics of the attachment of a fluorophore-labeled protein to a reaction partner bound to the surface of the cuvette were measured.
• Fig. 7 shows the change in emission against time.
• An increase in the emission (fluorescence counts) with the reaction time is observed, which corresponds to the attachment of the fluorophore-labeled protein to the reaction partner bound on the surface.

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