WO1993021530A1 - Procede et dispositif visant a augmenter la sensibilite et la selectivite pour les dosages immunologiques, les dosages d'interaction molecule-recepteur, adn complementaire - adn et molecule hote - molecule etrangere - Google Patents

Procede et dispositif visant a augmenter la sensibilite et la selectivite pour les dosages immunologiques, les dosages d'interaction molecule-recepteur, adn complementaire - adn et molecule hote - molecule etrangere Download PDF

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WO1993021530A1
WO1993021530A1 PCT/EP1992/001115 EP9201115W WO9321530A1 WO 1993021530 A1 WO1993021530 A1 WO 1993021530A1 EP 9201115 W EP9201115 W EP 9201115W WO 9321530 A1 WO9321530 A1 WO 9321530A1
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molecules
molecule
analyte
redox
measurement
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PCT/EP1992/001115
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German (de)
English (en)
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Karl Cammann
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Messerschmitt-Bölkow-Blohm Gmbh
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Publication of WO1993021530A1 publication Critical patent/WO1993021530A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the invention is directed to a generally applicable method consisting of a combination of selected steps and devices for extremely sensitive and undisturbed concentration determinations of any antibody-antigen pair, complementary molecule-receptor pairs, complementary DNA strands and selective guest / host molecule pairs.
  • the quantitative analysis of complex substance mixtures using the very selective antibody-antigen blinding (key-lock principle) and the DNA pair formation in the so-called DNA probes is an established analytical method in biochemistry and clinical chemistry and is accordingly widely used.
  • Competitive tests with labeled antigens, antibodies or DNA molecules are mostly used.
  • RIA radio immunoassay
  • the antigen added to the measurement solution (or the antibody molecule in the sandwich method) is radioactively marked.
  • EIA or the heterogeneous enzyme-linked adsorbend assay, ELISA enzyme immunoassay
  • ELISA enzyme immunoassay
  • labeled molecules compete with the unlabeled molecules to be measured (substance to be determined - analyte) for binding to the mostly carrier-bound antibody (or DNA sequence), so that the unknown amount of antigen (DNA type and amount) can be determined if for ver
  • a test with an antigen (DNA molecule) of known concentration is carried out immediately. With this method, the measurement signals are inversely proportional to the concentration.
  • Labeled antibodies are used in the so-called sandwich tests. These bind in a sandwich-like manner to antigen molecules, which in turn are previously bound to carrier-bound antibodies depending on the concentration.
  • the very specific binding of two complementary molecules also allows, conversely, the quantitative determination of the larger partner (antibody, receptor analysis or the complementary DNA molecule (or bio-surface), which marks a complementary DNA sequence in at least one part of the area Sequence) and are therefore the most important biochemical analysis methods.
  • antibody, receptor analysis or the complementary DNA molecule or bio-surface
  • these immunological methods are becoming increasingly important for environmental analysis.
  • the use of monoclonal antibodies for the various dioxins can significantly reduce the overall analysis costs by only having to carry out the very expensive GC-MS analysis (screening) if the immunological test is positive.
  • the advantageous structure of a simple and inexpensive electrochemical measuring system which requires the least amount of equipment in the form of potentiometry or amperometry, generally fails because the substance (analyte) to be determined is or is not present as a potential determining ion, but rather as a neutral molecule can be selectively implemented electrochemically.
  • optical methods spectrophotometry, fluorescence measurement, ellipsometry, surface plasmon resonance spectroscopy, evanescent wave method, etc.
  • Optical methods with the usual fluorescent markers which require an excitation wavelength in the UV or visible range, require a high level of equipment, which stands in the way of widespread routine use. The effort to stabilize the light source, to monochromatize using a stray light-free monochromator, to report the influence of extraneous light, to increase sensitivity, etc. requires equipment that is very complex and therefore expensive.
  • many substances are also stimulated in typical biological matrices (e.g.
  • German patent application DE 3916432 AI from 1990 describes a potentiometric method for the detection of an immuno-reaction between unlabeled complementary partners, but this method can also be used with smaller analyte molecules in a range below 1 ppm.
  • the object of the invention to increase the sensitivity while simultaneously reducing the known cross interference of all traditional immunoassays and similar methods is achieved in particular by the selected combination of several method steps according to claim 1.
  • the invention instead of the susceptible and inaccurate enzymatic signal amplification methods, repeatable electrochemical and optical measurement methods together with a special one electronic signal processing (signal medium value curve) and corresponding measuring arrangements.
  • the immuno-partner to be determined is either in the method according to the invention with a stable redox system or a stable one in the infrared range (> 700 nm) fluorescent compound durable (eg covalently bound).
  • a stable redox system or a stable one in the infrared range (> 700 nm) fluorescent compound durable (eg covalently bound).
  • the selection of these two marker molecule types was made after extensive preliminary tests and based on certain criteria, which are hereby disclosed.
  • Inorganic or organic systems with high standard exchange current densities on inert metal electrodes are preferably suitable as redox systems.
  • fluorescence markers the most advantageous marker molecules are molecules that can be excited with low-cost laser electrodes for fluorescence in the far infrared (> 800 nm).
  • the method according to the invention is simpler, more sensitive, faster, more correct and therefore ultimately also cheaper than all the immunoassays described so far.
  • the direct, extremely sensitive and rapid detection of smaller antigens is also more advantageous, which, according to the above-mentioned disclosure steps, is only poorly or not at all possible with the methods described there.
  • the labeled analyte molecule is previously bound to the immobilized partner.
  • the labeled analyte molecule is added to the sample in a known ratio and the mixture is allowed to interact with the immobilized immuno-partners (which include all of the complementary systems mentioned above). Depending on the concentration of the analyte, different amounts of the labeled analyte molecule are competitively bound, with an inverse relation.
  • the unbound or released labeled analyte molecules are again collected in a small space by means of the relevant immunological (complementary) partner, who is immobilized or localized and fixed, concentrated, and cleaned of adhering sample matrix by rinsing.
  • the device according to the invention in which there is an electrochemically and optically accessible surface, it is also possible to measure the part which does not correspond to the eluate collection.
  • the competitive test in which the surface of the device is covered with immobilized partner molecules which are saturated with your labeled complement molecule before contact with the sample, the decrease in the intensity of the label, which is proportional to the amount of analyte, can also be measured.
  • the other competitive test in which labeled and unlabeled analyte molecules are added together to the immobilized complement molecules that have free binding sites, the increase in labeled molecules in this surface area is inversely proportional to the concentration of the analyte.
  • the amount of labeled molecules that pass through the immunogenic zone unbound is directly proportional to the amount of analyte.
  • the analysis result can be obtained twice (redundantly) with such an immunological-analytical device.
  • This dual determination is new and significantly improves the reliability of the entire immunoassay. Errors and faults can be recognized immediately due to the mismatch between these two measured values.
  • the method according to the invention amounts to a double determination of the analyte.
  • the decrease in the signal intensity integrated over the immunogenic zone (proportional to the concentration) must correspond to the relevant increase in the "molecule trapping" zone (see FIG. 2) in a displacement assay.
  • the labeled analyte molecule is admixed in a known ratio before each analysis of the sample.
  • concentration of the labeled analyte molecules that can be measured there can be directly deduced from the concentration of the unlabeled analyte molecules that are present in the sample.
  • concentration of labeled analyte molecules after flowing through the immunogenic zone the higher the analyte concentration in the sample.
  • Another advantage (more sensitive and undisturbed than previous methods on a non-enzymatic basis) of the invention is the separation of the non-specifically bound matrix substances before the actual measurement and the collection or enrichment of the labeled molecules on a flat surface on which unlabeled partner molecules with free binding sites are fixed are bound so that they specifically bind the marked partners that have passed through the other immunological binding region unbound or have been released there (displaced from the binding) by the unmarked analyte.
  • the labeled analyte molecules bound on a flat surface can then be determined electrochemically or optically, depending on the type of labeling, both long measuring tents with high attenuation and Cyclic or repetitive measurements with electronic (or computer-aided) averaging are also possible. According to the laws of statistics, the signal-to-noise ratio can be improved by the factor root-N by means of N measurements.
  • a particularly simple and elegant device in connection with the invention is the use of test strip-like chromatographic material.
  • test strip-like chromatographic material both paper chromatography (expanded by membrane filter materials based on cellulose acetate, nitrate or the like) and thin-layer chromatography material suppliers (see Fig . 2).
  • the production of the devices according to the invention is very simple and efficient.
  • the corresponding poly- or monoclonal antibodies or F u fragments are correctly oriented using known binding techniques (covalent immobilization by means of spacer molecules and F c part-binding components) (binding sites to the outside) to more than 75 % of the test strip area applied.
  • this immunogenic zone corresponds to the size of the measuring range. It is then rinsed thoroughly to remove labeled analyte molecules bound by adsorption. The rest of the test strip now contains the analyte-recognizing antibody molecules (or in the other analogous reactions, the corresponding DNA's or host molecules) with free binding sites and serves as a molecule scavenger for the later measurement.
  • this Collection zone can advantageously be used a thin zone with dialysis properties.
  • the flowing base electrolyte (determined by the type of antibody molecules) can pass this zone, but the larger labeled analyte molecules are retained and thus also enriched in an extremely small zone.
  • This arrangement can also be carried out in transparent glass tubes or cheaper plastic tubes filled with the chromatographic material in question. If this tube is sealed with the dialyser membrane, other collecting zones can also be dispensed with. Here only the unimpeded access of the optical beam paths has to be guaranteed, which are preferably attached axially rather than to the side of the tube.
  • this dialysis film is attached so that it can be removed so that the inside of the planar electrochemical cell is made accessible to the labeled analyte molecules that have been released in the primary immunozone.
  • the latter can also be firmly applied to the inside of the dialysis filling beforehand using microelectronic methods.
  • the electrode surfaces are miniaturized and require only minimal amounts of precious metal, so that in this case the test tube-like structures can be discarded after a measurement. Because of the larger amount of immobilized analyte-recognizing and binding antibody molecules, the tube arrangement detects a larger concentration range of the analyte, i.e. the calibration curve changes to a saturation range later than with the planar arrangements.
  • the evaluation can also be carried out on the displaced routes (or integrated thereof) (see Fig. 2). An inexpensive laser diode scanner can be used for this.
  • a free end zone of the type described above can also be used Regulations serve, but in which the solvent evaporates.
  • the selected, stable redox marker molecules and the IR fluorescence molecules also tolerate temperature increases.
  • more easily evaporable solvents can also be used to increase efficiency, with addition being possible after the primary immunogenic zone. It is important that the evaporation to concentrate the marker molecules is limited to a small zone (area). Because the solvent transports the molecules to be detected there before it evaporates and leaves its transport freight there.
  • Fig. 1 to 2 illustrate the various arrangement options using sketches.
  • the electrochemical measuring method optimized according to the invention represents a cyclic voltammetrle in connection with a stable redox system, the latter being distinguished from all possible in that it has a particularly high standard exchange current density.
  • the cyclic voltammetrle guarantees with thin-film measuring cells that no measuring substance is converted electrochemically, ie is consumed. This is only oxidized and reduced.
  • the highest measurement sensitivity is achieved according to the invention when the potential range in which the working electrode (s) is operated cyclically includes the half-step potential of the redox marker (+/- 200-600 mV).
  • ferrocene blends ruthenium complexes, hexacyanoferrate II / III, J- / J etc.
  • ferrocene blends ruthenium complexes, hexacyanoferrate II / III, J- / J etc.
  • the best, reversible redox systems allow for more than 1000 cyclic voltammograms per second (1000 Hz!) Remaining stocks of less reversible, possibly disturbing redox systems from the sample matrix cannot be converted electrochemically so quickly and therefore do not generate a measurement signal (current flow).
  • other electrochemical methods can be used that do not lead to any consumption or net turnover of the reacting system, i.e. the method of pulse voltammetry or the differential pulse voltammetry or the square-wave voltammetry are also suitable for this, but cannot be done so quickly for fundamental reasons operate cyclically.
  • Another method step according to the invention which advantageously leads to the desired extreme increase in sensitivity, lies in a special compensation method for the capacitive current which increases at fast working electrode potential scan rates and forms the signal background.
  • a computer-controlled evaluation system which works like a scan recorder by means of a fast AD converter.
  • the compensation of the non-Faraday background current can be done in two ways, which can be used alone or together.
  • the channel assignment of the scan recorder is also reversed when the potential reversal point is reached, as if the voltage on the working electrode was recorded against the flowing current (C-V diagram) using an X-Y disc. Since this is equivalent to a simple signal addition, equally large anodic and cathodic currents are compensated for each measuring channel (tent window or potential). This is not the case only with the current peaks separated by approx. 60 mV.
  • a further step, which is significantly advantageous for the aim of this invention, is the electronic averaging that is only possible through rapid repeat measurements without consuming the measured substance.
  • the signal-to-noise ratio (S / R) can also be increased drastically, since several thousand cycles can be carried out. Since you can easily scan well over 100 potential cycles per second (»100 Hz) with reversible redox systems, there is an improvement in just one second, for example of the signal-to-noise ratio by a factor of 10.
  • improvements in this S / R ratio determining the detection limit result by several orders of magnitude, without reducing the sensitivity of the sensitive Protect protein molecules (enzymes) here.
  • the second method for suppressing the interfering capacitive current background in the cyclic voltammetrle uses an ultramicroelectrode array with individual electrode diameters of less than 10 mm as the working electrode. Anodic and cathodic current peaks no longer arise here, but a current stage with well-defined plateaus. Similar to polarography, the step height is proportional to the concentration of the electrochemically converted redox system (marker molecule).
  • the device according to the invention uses, for this purpose, a planar array arrangement of approximately 2000 individual electrodes with a diameter of 3 mm, in which the reference and counter electrodes are already integrated in the surface in an optimized manner. Only one of many possible geometrical arrangements has proven suitable here (see Fig. 3).
  • the new application of the cyclic voltammetrle to achieve extreme measuring sensitivity in redox systems or marker molecules with redox groups is possible because the alternating oxidation and reduction means that there is no consumption of the medium.
  • a path diffusion of the redox system is solved according to the invention in that a thin-film cell arrangement is selected.
  • the working electrode (s) and the counter and reference electrodes are pressed against the surface moistened with the base electrolyte with the redox-marked partner molecules.
  • the unlabeled partner molecules that bind the redox-labeled molecules can also be immobilized directly on or immediately next to the electrode surface.
  • the desired increase in sensitivity is obtained by combining several or at least two procedural measures.
  • the first measure is to use marker molecules that absorb strongly in the IR and fluoresce in the far infrared because the disruptive fluorescent background of some protein molecules (e.g. with biological matrices) does not occur and the inexpensive laser diodes are particularly advantageous as an excitation source with high spectral luminance. Due to its low beam convergence, the emitted laser light allows particularly precise delimitation of the measurement window.
  • fluorescent marker molecules are used, the fluorescence of which decays particularly slowly, so that this fluorescence can be easily separated from the rapidly decaying of the interfering molecules in the sample matrix.
  • the evaluation takes place at the Method according to the invention via a direct and faster measuring electronic consideration of the slower decay time.
  • a lock-in amplifier is used, which enables frequency and phase selective amplification.
  • the suppression of the disturbing fluorescence with the fast decay time is done here by a suitable choice of the phase angle on the measuring device.
  • fluorescence arrangement which excites by means of laser diodes to fluoresce at emission wavelengths> 700 nm and which quickly oscillates back and forth between an unlabeled background and the places where the optically marked analyte molecules are held on the relevant surfaces (alternatively, fluorescence markers can also be used, which is the method of delayed tenting
  • E an electronic signal averager or a computer-aided addition of signal-time curves (time proportional to Voltage during electrochemical detection or to the measuring location during the optical process). Statistical electronic noise is averaged to zero in the highest sensitivity ranges.
  • the surface with the immobilized immunological partner molecule for the analyte should be highly loaded in the interest of a large measuring range.
  • the molecule in question must also be immobilized there with a correct orientation (epitope area outside). This can also be accomplished, among other things, with the aid of a suitably chosen electrical field (or current) at the phase boundary of the carrier surface / measurement solution. This leads to a directed immobilization of these partners without the need for expensive special substances (as usual spacer molecules).
  • the directional immobilization together with a high immobilization density results in a high sensitivity and a large measuring range.
  • the immobilized partner molecules can also bind using the known techniques using the F c part
  • Spacer molecules in the immobilization of antibodies usual in environmental analysis are attached to support surfaces (e.g. glass beads, chromatographic stationary phases, microtiter plates etc.).
  • support surfaces e.g. glass beads, chromatographic stationary phases, microtiter plates etc.
  • paper also offers itself as an inexpensive carrier. Both laboratory filter paper and membrane filters based on cellulose or other materials can be used here. Chromatographic thin-film supports are also ideally suited for this purpose, since they can easily be modified on the surface.
  • a preferred type in this context are thin-layer plates with or without basic fluorescent marking, which are already in a so-called reversed Phase (RP material) were modified.
  • RP material reversed Phase
  • the partner of these mutually binding complementary molecules that is not the analyte in each case) succeeds particularly well if one or more long-chain aliphatic residues (C 6 -C 25 ) are synthesized specifically for these molecules in relation to the epitope region or in the case of antibodies, the genetically generated F ab fragments with the specific binding sites can generate lipophilic molecular groups at the opposite molecular site.
  • the lipophilic residues of the molecules to be immobilized are then immersed in the aliphatic molecular brush on the carrier surface and are thus fixed at the highest density without impairing the binding behavior. However, they can be eluted during denaturation or deactivation as is customary in chromatography, so that the support is available for immobilization with fresh molecules.
  • the latter can also be used elegantly as a counterelectrode in the electrochemical detection method.
  • This process can also be automated dynamically in the context of a flow injection analysis, ie the molecules to be immobilized (without bound analyte molecule with marker but also with) are first added to the unloaded RP surface before rinsing to remove the excess in the case of the unloaded molecules labeled analyte molecules are passed over the surface until saturation. After rinsing the sample again.
  • the redox systems selected in the invention are those which show the highest standard exchange current densities on the working electrode material used (platinum, gold, other noble metal). Of all possible redox systems, this includes preferably only the particularly reversible systems, such as those based on ferrocene, ruthenium complexes, hexacyanoferrate (II / III), iodine / iodide etc. Particularly advantageous redox systems have a redox potential close to that of oxygen, so that they are not disturbed by the latter and in this case the cyclic voltammetrle can also be carried out without inert gas flushing.
  • a planar microelectrode array arrangement can also be used particularly advantageously in the electrochemical cell, because the advantages of ultramicroelectrodes occur with single electrode diameters ⁇ 10 mm.
  • the latter are: redox stages instead of peaks in the voltammogram, reduction in the ratio of capacitive to Faraday current, stirring independence, quasi-consumption-free measurement without cycling the voltage, etc.
  • a particularly advantageous class of molecules for fluorescent labeling are, for example of bis-isothiocyanate derivative of
  • An advantageous embodiment of the arrangement and the method according to the invention is based on a competitive reaction between the labeled and unlabeled analyte molecule.
  • non-specific connections compensate each other, since they occur to the same extent in both cases.
  • the unlabeled analyte molecule have a slightly lower binding constant than the labeled one in order to be able to displace it more effectively. This is usually the case because the covalent linkage with a relatively large marker molecule weakens the antibody-antigen (or generally: molecule-complementary molecule) bond because both molecules can no longer approach each other optimally.
  • HSA human serum albumin
  • anti-HSA anti-HSA
  • BCPDA Europlum chelate
  • Several fluorescent chelate complexes could be covalently attached to an HSA macromolecule (20-30).
  • the europium chelate blend was used instead of the IR light-emitting marker, because the fluorescence that occurs here decays much more slowly than that of the interfering sample matrix (protein molecules in biological samples).
  • Measurements were taken at the end of a small column that was loaded with the Sepharose material and the marked immune complex.
  • light with a wavelength of 350 nm was used, the beam path of which was chopped by an optical chopper with a frequency of 140 Hz.
  • the fluorescent light was measured at right angles to the excitation light.
  • a photomultiplier was used as the light detector and a phase angle of 0 ° was set on the lock-in amplifier.
  • Fig. 5 shows the signals that can be measured by adding unlabeled HSA. They are proportional to the HSA concentration of the sample applied to the column.
  • the environmentally relevant atrazine was chosen as the analyte.
  • the latter was covalently linked to a modified Ferroeen molecule in 6 steps using standard methods of organic synthesis.
  • the selected, very reversible redox system had to be converted into a water-soluble form by adding strongly polar rare chains.
  • the individual chemical synthesis steps that were necessary - starting from the commercially available starting product - are shown in Fig.6.
  • Fig. 7 shows cyclic voltammograms of this redox-marked atrazine in extremely high dilution on a carrier surface. Due to the immediate Contact of the redox system with the working electrode surface and the absence of diffusing or diffusing redox molecules results in sharper peaks in the cyclic voltammogram than usual. This effect also increases the sensitivity.
  • Hexacyanoferrate (II / III) was chosen as the reversible redox system in a ratio of about 1: 1.
  • the typical detection limit for this system in the classic cyclic voltmeter lies in the millimolar concentration range.
  • the method of electronic signal averaging, which can be used here due to the consumption-free measurement technology, can, as Fig.8 shows, still extremely low redox concentrate ions far below the micromolar (mmol / L) range with a very good signal / noise ratio. At a cycle frequency of approx.
  • 1000 Hz for example, 1,000 oxidation and reduction reactions can be carried out in one second, which in terms of the electrochemical signal is equivalent to a ten thousand times higher redox content (compared to the usual 0.1 Hz catfish) or corresponds to a correspondingly increased number of electron transfers, which would be equivalent to an analyte molecule to which a corresponding number of one-electron redox molecules would be attached.
  • the surface of the working electrode is reduced, the associated double-layer capacity is reduced considerably more than the Faraday current density, ie the ratio of Faraday current (which is proportional to the analyte) to the capacitive is extremely improved, so that extremely fast potential change speeds of> 500 V / sec are possible.
  • the changed diffusion conditions cause the electroactive species to change their signal form.
  • ultra-microelectrodes (diameter ⁇ 10 mm) allow spherical diffusion conditions and convert so little substance that there is no depletion layer growing into the solution, which is indicated by a clear step and in the voltammogram stable limit current plateau shows. Fig.
  • FIG. 10 shows the difference between a cyclic voltammogram of a trace of a redox system with a macroelectrode and an ultra-microelectrode array of the geometric arrangement disclosed in Fig. 3.
  • the array has exactly the same properties as a single ultra-microelectrode, but has the advantage that the current strength is increased in accordance with the number of electrodes.
  • capillary forces can preferably also be used.
  • other supports can also be used for the immobilized partner molecules.
  • An example of this can be a piece of columnar chalk (or sintered glass / ceramic).
  • the antibody molecules are saturated with the labeled analyte molecules on the chalk surface before immobilization.
  • the immobilization can be done by simply immersing it in this solution while observing the running distance.
  • the molecule associates are cross-linked by means of bifunctional reagents or fixed to the surface. From this primary immunological zone, a second molecule-Sarnmel zone is established, separated by an untreated flow section.
  • the end surface with the glel Chen provided antibodies, which were also used in the primary immunological zone, with the difference that here the analyte-selective binding sites are free, so that they collect the labeled analyte molecules released by the unlabeled analyte in the sample and concentrate them in the smallest space.
  • the electrochemical or fluorometric measurement is then carried out on the polished end surface of the chalk column. The flow of a carrier electrolyte or the sample through the capillary forces is maintained by placing an absorbent plug on this end surface. The measured sample amount must be determined from the decrease in the sample volume.
  • a carrier for example flow-through column, flow paper, membrane filter etc.
  • a partner for example flow-through column, flow paper, membrane filter etc.
  • this is displaced by the unlabelled analyte by means of a competitive reaction.
  • it is bound again by immobilized complement molecules at the exit of the column before the measurement and thus enriched for the actual measurement, while at the same time interfering substances can also be removed by rinsing before the extremely sensitive electrochemical or optical measurement is repeated and the signal is added by generating the signal value -Noise ratio is increased arbitrarily.
  • a method for carrying out particularly sensitive immunoassays and other assays which are based on molecule-receptor, DNA-complementary-DNA strand and guest-host molecule interaction forces, characterized by rapid and repetitive measurements of the
  • Device characterized in that as the analyte-recognizing molecules or molecular associations the respective partners of the extremely selectively connecting molecule pairs: antibodies (antibody fragment with blinding site) - antigen, molecule - receptor, DNA section - complementary DNA section and Guest Molecule - Host molecule can be used immobilized on special surfaces and distributed in certain zones.
  • antibodies antibody fragment with blinding site
  • molecule - receptor DNA section - complementary DNA section
  • Guest Molecule - Host molecule can be used immobilized on special surfaces and distributed in certain zones.
  • Device characterized in that the partner molecule complementary to the analyte is immobilized on a stationary support at two separate zones, the molecules of the first zone being saturated at the beginning of an analysis with the redox- or fluorescence-labeled analyte molecule, so that thereby all binding sites are blocked and, when they come into contact with the unlabeled analyte molecule from the sample, the labeled analyte molecules are displaced, but the latter are collected again in the smallest possible volume elsewhere on the device before they are electrochemically or optically repetitive to the desired signal / noise ratio be measured.

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Abstract

Procédé permettant d'effectuer des dosages immunologiques particulièrement sensibles ainsi que d'autres dosages qui reposent sur des forces d'interaction molécule-récepteur, brin d'ADN complémentaire - brin d'ADN molécule hôte et molécule étrangère. Le procédé permet la réalisation de mesures rapides et répétitives du débit sanguin ou de la lumière fluorescente et deux mesures successives ainsi qu'une comparaison et une formation de quotient dans au moins deux zones de mesure ou étalonnage de manière similaire à la méthode de l'étalon interne, au moyen de molécules d'analytes stables marquées par fluorescence IR ou oxydoréduction, pour des immunodosages et d'autres analyses semblables.
PCT/EP1992/001115 1992-04-10 1992-05-20 Procede et dispositif visant a augmenter la sensibilite et la selectivite pour les dosages immunologiques, les dosages d'interaction molecule-recepteur, adn complementaire - adn et molecule hote - molecule etrangere WO1993021530A1 (fr)

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DE4212148 1992-04-10
DEP4212148.5 1992-04-10

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Cited By (5)

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WO1997036931A1 (fr) * 1996-04-01 1997-10-09 British Nuclear Fuels Plc Systeme de dosage et nouveaux composes marques utiles pour ce dosage
WO1999027368A1 (fr) * 1997-11-21 1999-06-03 Unilever Plc Ameliorations apportees aux analyses de deplacement
WO2003018834A2 (fr) 2001-08-25 2003-03-06 Friz Biochem Gmbh Essai de deplacement destine a la detection d'hybridations d'oligomeres d'acide nucleique
WO2003019194A2 (fr) * 2001-08-25 2003-03-06 Friz Biochem Gmbh Essai de deplacement pour la detection d'evenements d'association ligat-ligand
CN1296700C (zh) * 2003-12-31 2007-01-24 中国地质大学（武汉） 矿物材料红外荧光分析法

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DE19620636A1 (de) * 1995-06-01 1996-12-19 Fraunhofer Ges Forschung Vorrichtung zur Durchführung chemischer oder biochemischer Affinitätsverfahren und Verfahren zum Betreiben der Vorrichtung
DE19621165C1 (de) * 1996-05-24 1997-10-02 Karlsruhe Forschzent Verfahren zur Herstellung einer Probe aus immobilisierten Makromolekülen
DE19622458C2 (de) * 1996-05-24 1998-03-26 Senslab Ges Zur Entwicklung Un Enzymatisch-elektrochemischer Einschritt-Affinitätssensor zur quantitativen Bestimmung von Analyten in wäßrigen Medien und Affinitätsassay
DE19741716A1 (de) * 1997-09-22 1999-03-25 Hoechst Ag Adressierbares modulares Erkennungssystem, seine Herstellung und Verwendung
DE19742227A1 (de) * 1997-09-25 1999-04-01 Juergen Prof Dipl Phys Wolfrum Verfahren zum Sequenzieren eines einzelnen DNA-Moleküls
DE19964220C2 (de) 1998-11-23 2003-07-03 Friz Biochem Gmbh Verfahren zur Herstellung einer modifizierten leitfähigen Oberfläche
AU758063B2 (en) * 1999-01-18 2003-03-13 Friz Biochem Gesellschaft Fur Bioanalytik Mbh Method for electrochemically detecting nucleic acid-oligomer hybridisation events
DE10106654A1 (de) * 2001-02-12 2002-09-05 November Ag Molekulare Medizin Verfahren zum Nachweis und/oder zur Quantifizierung eines Analyten
US7341834B2 (en) 2003-12-15 2008-03-11 Geneohn Sciences, Inc. Multiplexed electrochemical detection system and method
WO2006065598A2 (fr) * 2004-12-13 2006-06-22 Geneohm Sciences, Inc. Cartouches fluidiques utilisees dans la detection electrochimique de l'adn

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EP0046004A1 (fr) * 1980-08-07 1982-02-17 Syva Company Méthode de concentration à zone dans les immuno-essais hétérogènes
EP0171148A1 (fr) * 1984-06-13 1986-02-12 ARES-SERONO RESEARCH & DEVELOPMENT LIMITED PARTNERSHIP Dispositifs utilisés pour des méthodes d'analyse chimique
US4859583A (en) * 1985-02-25 1989-08-22 Amoco Corporation Chemiluminescent immunochemical technique for low molecular weight antigens
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EP0201339A2 (fr) * 1985-05-09 1986-11-12 Ultra Diagnostic Corporation Méthode d'essai immunologique, dispositif et trousse de réactifs

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997036931A1 (fr) * 1996-04-01 1997-10-09 British Nuclear Fuels Plc Systeme de dosage et nouveaux composes marques utiles pour ce dosage
US6576475B1 (en) 1996-04-01 2003-06-10 University Of Liverpool Assay system and novel labelled compounds for use therewith
WO1999027368A1 (fr) * 1997-11-21 1999-06-03 Unilever Plc Ameliorations apportees aux analyses de deplacement
WO2003018834A2 (fr) 2001-08-25 2003-03-06 Friz Biochem Gmbh Essai de deplacement destine a la detection d'hybridations d'oligomeres d'acide nucleique
WO2003019194A2 (fr) * 2001-08-25 2003-03-06 Friz Biochem Gmbh Essai de deplacement pour la detection d'evenements d'association ligat-ligand
WO2003018834A3 (fr) * 2001-08-25 2003-09-12 Friz Biochem Gmbh Essai de deplacement destine a la detection d'hybridations d'oligomeres d'acide nucleique
WO2003019194A3 (fr) * 2001-08-25 2004-01-29 Friz Biochem Gmbh Essai de deplacement pour la detection d'evenements d'association ligat-ligand
CN1296700C (zh) * 2003-12-31 2007-01-24 中国地质大学（武汉） 矿物材料红外荧光分析法

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DE4216696C2 (de) 1995-01-26

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