WO1993021530A1

WO1993021530A1 - PROCESS AND DEVICE FOR INCREASING THE SENSITIVITY AND SELECTIVITY OF IMMUNO-ASSAYS, MOLECULE-RECEPTOR, DNA-COMPLEMENTARY DNA and FOREIGN MOLECULE-HOST MOLECULE INTERACTION ASSAYS

Info

Publication number: WO1993021530A1
Application number: PCT/EP1992/001115
Authority: WO
Inventors: Karl Cammann
Original assignee: Messerschmitt-Bölkow-Blohm Gmbh
Priority date: 1992-04-10
Filing date: 1992-05-20
Publication date: 1993-10-28
Also published as: DE4216696C2; DE4216696A1

Abstract

A process is disclosed for carrying out particularly sensitive immuo-assays and other assays that rely on molecule-receptor, DNA strand-complementary DNA strand and foreign molecule-host molecule interaction forces, with rapid and repetitive measurements of the current density or fluorescent light and double measurement and comparison/quotient calculation in at least two measurement zones or calibration in an analogue manner to the inner standard method, by using stable redox or IR-fluorescence marked analyte molecules in immuno-assays and the like.

Description

Method and device for increasing sensitivity and selectivity in immunoassays, molecule receptor, DNA complementary DNA and guest-host molecule interaction assays

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.

It also relates to a device for sensitivity and

Selectivity increase in immunoassays, molecule receptor,

DNA Complementary DNA and Guest Host Molecule Interaction Assays.

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. In the radio immunoassay (RIA), the antigen added to the measurement solution (or the antibody molecule in the sandwich method) is radioactively marked. In the enzyme immunoassay (EIA or the heterogeneous enzyme-linked adsorbend assay, ELISA), a so-called marker enzyme is bound to the marker molecules in question. These 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.

Conversely, 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. Now that it is possible to produce monoclonal antibodies in any quantity for smaller molecules (haptens), these immunological methods are becoming increasingly important for environmental analysis. For example, 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 now traditional immunological methods of determination (RIA and EIA or ELISA) have considerable night-time effects, which are well known. Either disposal, shipping and storage problems arise due to the use of radioactive materials or one has to reckon with an influence on the immunological reaction by the enzyme molecule, which is usually voluminous compared to the relatively small antigen molecule. Enzyme-labeled antigen or antibody molecules (or one of the partners of molecule-receptor, guest-host molecule, DNA-complementary DNA interaction methods) have all the disadvantages of enzymatic processes. The life span of the enzyme is limited and the enzyme reaction (biocatalysis) can be sensitively disturbed by unknown environmental matrices by inhibitors or enzyme poisons (eg heavy metals), so that incorrect results occur. In most cases, the enzyme-labeled compound must be stored in a cool place for reasons of stability. Because of this diminishing activity additional calibration steps are required and the GLP guidelines (good laboratory practice), which must be observed in quantitative analyzes, require a lot of the more expensive enzyme-labeled compounds by means of reserve samples. Phase separation is also required, a substrate must be added, which makes handling very complicated. The necessary work steps usually take a few hours. The so-called homogeneous enzyme immunoassays (EMIT) run without phase separation. They work competitively and are only suitable for small antigen molecules. There is only little preliminary work on a simple and generally applicable analytical-chemical exploitation of selective molecule-receptor interaction forces, DNA pairing reactions and the equally selective guest-host relationships of supramolecular chemistry for the purpose of quantitative determination or even to set up appropriate sensors.

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. The use of optical methods (spectrophotometry, fluorescence measurement, ellipsometry, surface plasmon resonance spectroscopy, evanescent wave method, etc.) for the detection of the above-mentioned specific molecular interactions has been described. Some can detect the immunological reaction even without a marked partner due to the change in the refractive index at a phase boundary, but have major problems with small analyte molecules (as is common in environmental analysis) in the trace range (<1 ppm) because the non-specific binding of interfering accompanying substances is usually present in a large excess in addition to the analyte in a real sample, is sensitivity-limiting. 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. In addition to the fluorescent molecular groups of the marker, many substances are also stimulated in typical biological matrices (e.g.

Tryptophan and others) so that their fluorescence cannot be distinguished from that of the marker molecule, which extremely worsens the detection limit. Only the marking using special fluorescence markers, which work according to the time-delayed fluorescence method, can avoid these problems. However, this method requires more expensive devices and is practically only carried out using so-called enhancement steps to increase the sensitivity, but this means an additional work cycle.

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.

In the case of immunoassays which are operated like affinity chromatography and in which a displacement reaction with a labeled analyte molecule is used, the prior art has only described measurements by means of flow measuring cells in which the interaction time (= pure measuring time) with the measuring variables (light, Current etc.), given by the flow velocity, is relatively short (only a few seconds). Such a short measuring time does not allow high electronic damping, especially for flow measurements. Therefore, the electronic or optoelectronic amplification technology is due to the signal-to-noise ratio, which drops sharply in the most sensitive areas

(S / R ratio) set limits. Are also impossible when the marked substance flows quickly through the flow de Repetitive measurements that have a similarly positive effect on the S / R ratio when averaging. In addition, repeat measurements require a reference point, ie a background measurement (blank value measurement or blanc value), which requires a corresponding device. There is nothing reported in the prior art about a simple and quasi-automatic separation of the sample matrix with disruptive components also emerging from the immunological reaction zone, except in connection with the heterogeneous ELI-SA techniques which prescribe an additional separation step. However, this is of central importance for correct measurements and an indispensable prerequisite for repetitive measurements, in which incorrect measurements naturally also add up.

Equal treatment of immunoreactions with receptor reactions or DNA pairing and molecule-recognizing guest / host molecule incorporations with regard to the generally applicable, sensor-utilizable arrangement according to the invention has not been described so far, since labeling with a voluminous enzyme very strongly hinders these selective binding reactions (steric disability ). This is not the case with the relatively small redox systems or IR fluorescent molecules.

There is therefore a need for a generally applicable method and a device which reports the known night-time results of the methods which display directly and also those which are carried out with labeled immunological partners (all current immunoassays).

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. In order to achieve the required extremely low detection limit in the trace analysis range, 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 (antigen or antibody, molecule or associated receptor, guest molecule or associated host molecule, DNA strand and associated complementary sequence) 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). 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. With 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. Here, however, there is a large area of application for new molecular analysis (pollutant analysis), especially in the area of environmental protection or monitoring of maximum workplace concentrations as well as clinical chemistry (medical diagnosis, pathology, forensic medicine).

The method according to the invention can be carried out in different ways:

In an advantageous version, the labeled analyte molecule is previously bound to the immobilized partner. The unlabeled analyte molecule in the sample displaces its labeled counterpart (redox- or fluorescence-labeled antigen or antibody molecule = labeled analyte molecule) from the surface upon contact with the bound, labeled partners Chenbinding, which is then transported away by a liquid flow.

In another version, 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. In both versions, 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.

In 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. In 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. In 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. Here the amount of labeled molecules that pass through the immunogenic zone unbound is directly proportional to the amount of analyte. In the case of such accessible surfaces, 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 same applies analogously to the method in which the labeled analyte molecule is admixed in a known ratio before each analysis of the sample. By collecting the unbound labeled and unlabeled analyte molecules in a special capture zone, the 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. The higher the 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, the amount of which, according to calibration, allows information about the (unlabeled) analyte molecules present in the sample, 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.

Instead of the specifically binding immuno-partner molecules with free binding sites as selective "molecule scavengers" after the primary competitive immuno-reaction, other collection phases can also be used. In the case of lipophilic analyte molecules, a zone to be flowed through which is impregnated with oil or wax (or PVC plus plasticizer or the like) is sufficient. Alternatively, a material can be used for this, which is common in reversed phase chromatography (eg RP-18 stationary phase). The latter is particularly useful when ionic analyte molecules are to be retained. Here you add an appropriately charged, lipophilic counterion to the flow solution. This leads to ion pair blinding in the oil or fat, which makes the passage of the labeled analyte molecules through this molecule trapping zone more difficult. With a flowing carrier solution, they will accumulate towards the end of the lipohi zone and can thus be made accessible to the sensitive measurement.

A particularly simple and elegant device in connection with the invention is the use of test strip-like chromatographic material. Here, 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. In the field of environmental analytical application of the method disclosed here, 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. After fixing these analyte-recognizing macromolecules on the test strip surface, the initial area is immersed in a solution that contains only labeled analyte molecules. As a result, all binding sites are saturated with this labeled analyte molecule. The length of 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. To conclude this Collection zone can advantageously be used a thin zone with dialysis properties.

It is thereby achieved that 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. In the case of redox-labeled analyte molecules, 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. Of course, 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.

To enrich the released or unbound labeled analyte molecules, both in the displacement method and in the competitive method (adding labeled analyte molecules at the beginning of the assay), 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. In the case of antibody compatibility, 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). At high potential change rates (»500 mV / sec scan rate), working electrodes> 10 mm produce anodic and cathodic current peaks which are proportional to the concentration of the redox system. The higher the scan rate, the greater the current peaks, which equates to an increase in sensitivity. If systems with the highest standard exchange current densities (e.g. ferrocene blends, ruthenium complexes, hexacyanoferrate II / III, J- / J etc.) are used as redox markers, then not completely separated redox systems from the sample matrix interfere significantly less when working with short cycle tents (faster voltage ramps) 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). . Alternatively, 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.

For this purpose, a computer-controlled evaluation system is used, which works like a scan recorder by means of a fast AD converter. The compensation of the non-Faraday background current, which also contributes to the increase in sensitivity, can be done in two ways, which can be used alone or together. In the case of working electrodes with a diameter of more than 10 mm, 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. With a signal averager, 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. In measuring tents which correspond to those of the immunoassay method with enzyme markers, 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. Here, 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. If necessary, the unlabeled partner molecules that bind the redox-labeled molecules can also be immobilized directly on or immediately next to the electrode surface. The cyclization is necessary at detection limits below 1 ppb (redox system), because at such extremely low concentrations, all molecules must be detected electrochemically if possible. Then a current of one mA corresponds to a converted amount of substance of approx. 10 mol / sec. Good electrochemical measuring cells currently also allow reliable current measurements in the nA range, which corresponds to a conversion rate that is a thousand times smaller. By the signal averaging is now measurable pico and femto ampere currents, which leads to conversion rates of 10 - ¹⁶ resp. 10- ¹⁹ mol / sec leads.

In optical detection, 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. As a further measure, 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. In contrast to a commercially available arrangement, in which the excitation light is simply prevented by a shutter, and the actual measurement of the fluorescent light only begins after a few milliseconds (after the interference fluorescence radiation has completely decayed) and is integrated over a certain tent, the evaluation takes place at the Method according to the invention via a direct and faster measuring electronic consideration of the slower decay time. For this purpose, 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. The advantage of this method compared to the counting rate method (photon counting) of the commercial version is the higher sensitivity, because the frequency-selective amplification amplifies only a fraction of the mostly white electronic noise of the photo receiver and because it is possible to work at higher light chopper frequencies, which drastically increases the display speed increased, so that flow measurements, as usual with the FIA, can still be carried out with corresponding sensitivity. These advantages are obtained in the invention by:

A) a kind of affinity chromatographic column or also an open or surface of any carrier material with immobilized partner molecules to the molecule to be determined (analyte) that is accessible to optical and electrochemical measurement;

B) a similar "Sarnmel surface" accessible to the measurement for released labeled analyte molecules (in the event of a displacement reaction on the above column or surface which is covered as completely as possible with labeled immunogen-bound analyte molecules by unlabeled analyte molecules);

C) a flat electrochemical measuring cell according to the invention in a potentiostatic circuit consisting of two or three electrodes with one or more working electrodes, which carries out a cyclic voltammetry in the electroactive area of the redox system in question (repetitive oxidation and reduction) and which is in direct contact with the "surface of the electrode" or that can be brought under A);

D) a 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

Enable fluorescence, but here the direct and fast measurement takes place by means of an electronic phase-selective amplifier, which can also oscillate quickly between an unmarked and marked surface with the help of light guides).

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.

F) If both the change in the labeled analyte molecules in the primary immunogenic zone and in the Sarnmel zone is detected during the evaluative measurement, a kind of internal standardization can be carried out in addition to a double measurement by forming quotients. This improves the reliability of the measurements similar to the internal standardization of emission spectroscopic methods.

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. Alternatively, 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.).

As a special variant of the exemplary embodiments, 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. Here, the directed immobilization of the partner molecule type (i.e. 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. When using thin-layer plates based on an aluminum foil, 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 use of accessible flat surfaces for the immobilization of the partner molecules can save one procedural step, because the amount of the labeled analyte molecules bound there can be used in both versions (pre-immobilization with labeled analyte molecules and competition from the labeled analyte molecules added to the sample) with both the planar electrochemical Easily determine the measuring cell (simply by pressing it onto the surface) or with the optical fluorescence method without using an additional Sarnmel surface. With both detection methods, it is important that interfering substances are rinsed away from the sample matrix before the actual measurements. This is done using pure, buffered electrolyte solution, which both supports the stability of the biomolecules and also represents the basic electrolyte for the electrochemical or optical type of detection.

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

2- [4'-chloro-7 '(3 "-ethyl-2" -benzothiazolinyliden) -3', 5 '- (1'"- propanediy 1) -1 ', 3', 5'-heptatrlen-1 ' -yl] -3-ethylbenzothiazoliumbromid large porphyrin rings or suitable phthalocyanines (see Fig. 4). They can be excited by the inexpensive long-wave monochromatic and extraneous light-free laser light sources and generate fluorescence in a wavelength range where hardly any other molecules interfere. The use of laser electrodes With corresponding IR-sensitive semiconductor detectors, particularly small measuring setups (eg hand-held measuring devices) are possible. The devices and working methods according to the invention are explained in more detail with reference to examples:

Example 1

Displacement of the labeled analyte molecule from the immunologically prepared surface area by the unlabeled analyte molecule in the sample:

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. Here, non-specific connections compensate each other, since they occur to the same extent in both cases. It is also desirable that 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.

In this example, human serum albumin (HSA) was chosen as the model analyte. The associated antibody (anti-HSA) was used as the immobilized partner molecule. The anti-HSA antibodies were attached to the surface of Sepharose material with a small grain size (150 mm) using the known immobilization techniques and coated with fluorescence-labeled HSA. A Europlum chelate (BCPDA) was chosen as the fluorescence molecule. Several fluorescent chelate complexes could be covalently attached to an HSA macromolecule (20-30). In this example, 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. When adding unlabelled HSA, this was provided with the fluorescent marker ne measured HSA molecule displaced from the surface of the Sepharose particles loaded with HSA antibodies in a flow measuring cell. For this, 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. With this measuring technique, significantly more samples can be analyzed per unit of time than with the photon count, which also remains closed to the FIA technique. In addition to HSA, other analytes such as atrazine or other pollutants could also be determined very sensitively in the sub ppm range with high accuracy by immobilizing the corresponding mono- and polyclonal antibodies.

Example 2

Electrochemical detection of redox-labeled analyte molecules:

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.

The last compound, the redox-labeled atrazine, was used in an atrazine assay after purification both for the displacement method and for the competitive method. The corresponding antibodies were by Hock et al. described in the literature [1,2]. 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.

Example 3

Increasing the detection limit by repetitive electrochemical oxidation with subsequent reduction:

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.

Example 4

Suppression of the capacitive current component in fast cyclic voltammograms by using the cyclical averaging: As is well known, a so-called capacitive current occurs at rapid potential change rates due to the corresponding charge reversal of the double layer capacitance at the working electrode interface, the size of which is unfortunately proportional to the rate of change, so that an extremely fast cycle speed should actually be ruled out in the cyclic voltammetrle, because of this proportion of the relevant redox peak (Faraday current) by far and leads to voltammograms, as shown in Fig. 9. On the other hand, if the voltage coordinate values for macro working electrodes are counted downwards on the individual digital channels of the averaging device or scan recorder using this device, similar to an XY recorder, the reversal of the sign of the current strength during addition leads to one and the same channel (the is assigned to a precisely determined working electrode potential) for compensation of the capacitive current component. This happens almost completely, since the capacitance current with a constant double layer capacitance (with the same working electrode potential) is proportional to the sign of the potential change rate dU / dt. Only the Faraday current peaks, which are proportional to the concentration of the redox system, are not compensated for by this circuit trick, since they run at different potentials. So you get a low noise, averaged cyclic voltammogram that looks as shown in Fig.8e. It is only through this circuitry and electronic device that it is possible to work with potential cycle rates of well over 100 Hz.

Example 5

Suppression of the capacitive current component by using an ultra microelectrode array:

If 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. In addition, the changed diffusion conditions cause the electroactive species to change their signal form. In contrast to planar diffusion in macroelectrodes, 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. 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.

Example 6

Arrangement for a particularly simple embodiment:

Instead of the forced flow in the flow injection analysis, capillary forces can preferably also be used. In addition to the principle of paper and thin layer chromatography, 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). In hapten-like analytes, 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. For reasons of high sensitivity, a narrow area directly at the end of the chalk support is sufficient. In the example, 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.

If a carrier (for example flow-through column, flow paper, membrane filter etc.) is used in the invention, to which a partner is bound, this is displaced by the unlabelled analyte by means of a competitive reaction. However, 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.

Claims

1. 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

Current strength or fluorescent light and double measurement and comparison / quotient formation in at least two measuring zones or calibration analogous to the method of the internal standard, using stable redox- or IR-fluorescence-labeled analyte molecules in immunoassays and similar assays.

2. Device according to claim 1, 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.

3. Device according to claim 2, 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.

Claims

4. Apparatus according to claim 2 and 3, characterized in that in addition to the displacement reaction, a competitive binding reaction between the unlabeled analyte molecule in a sample and the redox- or fluorescence-labeled analyte molecule, which must be added to each sample in a known ratio, can take place on carrier-bound partner molecules , whereby after rinsing the unbound molecules and removing the sample matrix, the repetitive electrochemical or optical measurement can take place.

5. Apparatus according to claim 2 to 4, characterized in that the analyte-recognizing complement molecules in question are immobilized on support surfaces which enable repetitive electrochemical or optical measurement, ie either allow intimate contact with a planar electrochemical three-electrode measuring cell or the measurement of the IR fluorescent light.

6. Apparatus according to claim 2 to 5, characterized in that preferably chromatographic stationary phases, such as adsorbent-filled columns, RP-18-like materials, RP-18-like materials, flow paper strips, membrane filter strips, as a carrier for the analyte-recognizing and selectively binding complement molecules fixed at certain points Cellulose bases or the like which have capillary flow properties can be used.

7. The device according to claim 2 to 6, characterized in that on a sample run (type affinity chromatographic miniature column) at least one primary immunological (or generally: analyte-specific binding) zone is optionally present together with another Sarnmel and enrichment zone.

8. The device according to claim 7, characterized in that the affinity chromatography separation, displacement or competitive connection is carried out either planar, so that the planar electrochemical three-electrode electrode can find unimpeded contact with different surface sections and also the optical measurement alternatively between a substrate and the measuring zones can oscillate.

9. The device according to claim 7, characterized in that the Sarnmel and enrichment phase, narrowly defined zones in the sample flow direction behind the primary immunogenic zone (above definition) are created, which consist of the relevant analyte-recognizing and binding complement molecules, but which are free Have binding sites and lead the redox-active or fluorescent analyte molecules collected in this way to the repetitive measurement.

10. The device according to claim 9, characterized in that the second Sarnmel and enrichment zone, the lipophilicity of the labeled analyte molecules is exploited in that a lipophilic zone with an oil- or wax-soaked surface or RP-18 material is used instead of the complement molecules in question, with ionic analytes of this phase a lipophilic counter ion is added to form the ion pair.

11. The device according to claim 9, characterized in that the Sarnmel and enrichment phase for the labeled analyte molecules displaced in the primary reaction zone is given by a dialysis membrane selected according to the molecular weight of the labeled analyte molecule, which is attached at a certain distance from the primary reaction zone, and which can be measured electrochemically both stationary using tapped electrodes and dismantled.

12. The apparatus according to claim 11, characterized in that this dialysis membrane additionally contains the analyte-binding complementary molecules immobilized on the surface lying first in the direction of flow.

13. The apparatus according to claim 9, characterized in that the Sarnmel and enrichment zone is achieved in that the solvent is evaporated by the application of heat and transport gas at the end of the sample run.

14. The apparatus according to claim 2 to 13, characterized in that the primary analyzer-recognizing and binding reaction zone and the separate Sarnmel and enrichment zone is within a test strip-like carrier with capillary forces, so that the transport of the liquid sample through the different zones is achieved by the latter and an external pump is not necessary, the evaluation taking place over concentration-proportional routes or integral signals over the characteristic route sections and is also redundant. The evaluation device is a scanner with optical or electrical measurement. Roller-shaped working electrodes are used in the electrochemical "scanning".

15. The device according to claim 2 to 14, characterized in that the measurement of the concentration of the redox-labeled analyte molecules on the corresponding surface zones of the device according to the invention by the electrochemical method of rapid cyclic voltammetry or another method that works without net conversion (substance consumption), by means of a planar three-electrode measuring cell, which is pressed onto these points together with a suitable base electrolyte (principle of the thin-film measuring cell).

16. The apparatus according to claim 15, characterized in that the planar or quasi-planar electrochemical three-electrode measuring cell instead of a macro working electrode made of a noble metal such as platinum, gold or the like. contains an ultra-microelectrode array made of the same materials with individual electrode diameters <10 mm in order to reduce the capacitive current that rises sharply with fast cyclic voltammograms.

17. The apparatus of claim 15 and 16, characterized in that the working electrode or the ultra-microelectrode array used for this purpose are integrated into the relevant zones of the assay.

18. The apparatus according to claim 15 to 17, characterized in that the cyclic voltammogram with a high repetition rate in loading range 1 to »100 Hz is not registered as usual on an XY recorder but on an electronic averager that is switched so that the digitized current signals in both potential directions are added to one and the same voltage-proportional channel, which is due to the different sign of the capacitive Current share in the different directions of change equals almost 100% compensation of this disruptive non-Faraday current, while the concentration-dependent current peaks of the redox molecules occur at different potentials and therefore do not compensate each other.

19. The apparatus according to claim 18, characterized in that the ultra-microelectrode array is used as the working electrode.

20. The apparatus according to claim 15 to 19, characterized in that the measuring process of an analyte follows automatically computer-controlled in the catfish, that both the amount of sample taken up by the arrangement and the Sarnmelzeit are reproducibly observed and the repetitive electrochemical measurement with the same number of addition cycles , which depends on the signal / noise ratio, is carried out.

21. The apparatus according to claim 20, characterized in that redox systems with a particularly high standard exchange current density are used as redox marker molecules which are covalently linked to the analyte molecule, which dissolve in the solvent in question or in the sample matrix.

22. The apparatus according to claim 21, characterized in that as particularly reversible redox systems water-soluble, stable and storable ferrocene derivatives, ruthenium complexes, hydrochlorones, hexacyanoferrate (II / III), iodine / iodide or the like. are used, which are connected to the analyte molecule via spacer molecules.

23. The device according to claim 2 to 14, characterized in that the marker molecules for repetitive optical detection stable, camp capable special molecules are used, which absorb light of the wavelength> 700 nm and emit again in the long-wave range as fluorescent light.

24. The apparatus according to claim 23, characterized in that the bis-isothiocyanate derivative of 2- [4'-chloro-7 '(3 "-ethyl-2" -benzothiazolinylidene) - 3' as the molecular class for the advantageous IR fluorescence labeling , 5 '- (1' "- propanedyl) -1 ', 3', 5 '-heptatrien-1' yl] -3-ethylbenzothiazolium bromide, porphyrin derivatives with large 2n + 1 Pi electron systems or phthalocyanines, which can also be used distinguish particularly high extinction coefficients.

25. The device according to claim 2 to 14, characterized in that the repetitive optical measurement, which is supplied to the averager is achieved by alternately measuring the fluorescent surface zone to be measured and the substrate at a point not covered with the Sarnmel phase or the complementary molecules , which can be achieved by a relative movement of the optical beam.

26. The apparatus of claim 2 to 14, characterized in that an intermittently operated laser diode in the IR wavelength range with high radiation density, monochromaticity and freedom from stray light is used together with a lock-in amplification technique as an optical excitation source instead of a white light source and a monochromator.

27. Method for determining the concentration of a partner of a chemically selectively recognizing complementary pair of molecules (antibody-antigen, receptor-receptor molecule, DNA sequence-complementary

DNA strand, guest-host molecule) by means of an immunoassay according to claims 1 to 26, characterized in that redox- and fluorescence-labeled analyte molecules are used and the electrochemical or optical measurement of the distribution of these labeled molecules in the different zones of the test device without Material consumption takes place so that any number of repetitive measurements are possible.

28. The method according to claim 1 and 27, characterized in that the repetitive measurements are reproducibly added to a signal mean, so that the signal / noise ratio between the Faraday current and the background and the surface with the fluorescent molecules and the free carrier surface is significantly improved.

Summary:

Process 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, with rapid and repetitive measurements of the current intensity or fluorescent light and double measurement and comparison / Quotient formation in at least two measuring zones or calibration analogous to the method of the internal standard, using stable redox- or IR-fluorescence-labeled analyte molecules in immunoassays and similar assays.

Fig. 1