DE4216696A1 - Measurement of complementary interaction, e.g. in immunoassays - comprising measurement of fluorescence or redox (current) strength with double measurements and comparisons of at least two measuring zones - Google Patents

Abstract

Claimed is (A) a sensitive method for immunoassays or assays based on complementary interaction by (a) quick and repetitive measurements of electric current or fluorescence, and (b) double measurements either in at least two measuring zones or by internal calibration of the system using stable redox or IR-fluorescence-labelled analytes followed by comparison of the two measurements obtd.. Also claimed is an appts. for performing (A) wherein the analytes recognising complementary mols. (antibodies (ABs), receptors, DNA-fragments) are immobilised at special surfaces. USE/ADVANTAGE - The new method is used in the detection of binding pairs such as in immunoassays. This method is simpler, faster, more correct, cheaper and more sensitive than known methods such as ELISAs RIAs or EIAs.

Description

The invention is directed to a generally applicable method ren, which consists of a combination of selected steps and Devices for extremely sensitive and undisturbed concentra complementary determination determinations of any antibody-antigen pairs rer molecule-receptor pairs, complementary DNA strands and selek tive guest / host molecule pairs. It also concerns a pre direction to increase sensitivity and selectivity 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 binding (key-lock-prin zip) and the DNA pair formation in the so-called DNA probes is in an established analysis of biochemistry and clinical chemistry method and accordingly widely used. Most are competi tive tests with labeled antigens, antibodies or DNA molecules len used. In the radio immunoassay (RIA), this is the measurement solution added antigen (or the antibody molecule in the sandwich Method) radioactively marked. In the enzyme immunoassay (EIA or the heterogeneous enzyme-linked adsorbend assay (ELISA) is to the a so-called marker enzyme bound. These labeled molecules compete with the unlabeled ones to be measured molecules (substance to be determined = analyte) to bind the mostly carrier-bound antibody (or DNA sequence), so that determine the unknown amount of antigen (DNA type and amount)  if, for comparison, a test with an antigen (DNA molecule) known concentration is carried out. The measurement signals are at this method is inversely proportional to concentration. Both So-called sandwich tests, labeled antibodies are used. This am the sandwiched to antigen molecules, which in turn previously con are bound to carrier-bound antibodies depending on the concentration.

The very specific binding of two complementary molecules conversely, of course, also allows the quantitative determination of the larger partners (antibody, receptor analysis or comple mental DNA molecule (or bio-surface), which is at least in a partial area a complementary DNA sequence to the labeled Sequence) and are therefore the most important biochemical Analytical method. Now that it is possible, also for smaller molecules (haptens) monoclonal antibodies in any These immunological methods will also produce quantities increasingly important for environmental analysis. For example one using monoclonal antibodies for the various Dioxins significantly reduce the total analysis costs in that only if the result of the immunological test is positive expensive GC-MS analysis must be carried out (screening).

The now traditional immunological methods of determination (RIA and EIA or ELISA) have considerable disadvantages are well known. Either kick radio because of the use active material disposal, shipping and storage problems or one has to influence the immunological reaction mostly because compared to the relatively small antigen molecule, voluminous enzyme 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 lifespan of the enzyme is limited and the enzymatic reaction (Biocatalysis) can be done with unknown environmental matrices by Inhibito or enzyme poisons (e.g. heavy metals) the so that wrong results occur. Usually the enzymmar Kiert connection be stored cool for stability reasons. Because of this decreasing activity there are additional calibrations steps required and the GLP guidelines (good laboratory practice),  that have to be observed in quantitative analyzes retained a lot of the more expensive enzyme-labeled samples Links. A phase separation is also required, a sub strat must be added, which makes handling very complicated is decorated. The necessary work steps usually require some hours. The so-called homogeneous run without phase separation Enzyme immunoassays (EMIT) that work competitively and themselves only suitable for small antigen molecules.

Via a simple and generally applicable analytical-chemical Exploitation of selective molecular receptor interaction forces, DNA pairing reactions as well as the equally selective guest host relationships of supramolecular chemistry for the purpose of a quantitative determination or even to build up corresponding Sensors do little preliminary work.

The advantageous structure of a simple and inexpensive electro chemical measuring system, in the form of potentiometry or ampero requires the least amount of equipment, fails in the rule that the substance to be determined (analyte) is not as potential determining ion, but as a neutral molecule or can not be selectively implemented electrochemically. The Use of optical methods (spectrophotometry, fluorescence measurement, ellipsometry, surface plasmon resonance spectroscopy, Evanescent wave method, etc.) to capture the above The specific molecular interactions mentioned are described been. Some can see the immunological response without mar selected partners due to the change in the refractive index a phase boundary, but have big problems with small ones Analyte molecules (as usual in environmental analysis) in the trace range (<1 ppm) because the non-specific binding of disruptive Accompanying substances, which are even mostly mostly in a large excess the analyte is present in a real sample is limiting.

Optical methods with the usual fluorescent markers, the one Require excitation wavelength in the UV or visible range, need a lot of equipment, which is widespread routine application is in the way. The effort to stabilize  tion of the light source, for monochromatization using a scatter light-free monochromator, to avoid extraneous light influences sen, to increase sensitivity, etc. requires equipment buildings that are very complex and therefore expensive. Also be besides the fluorescent molecular groups of the marker also many Substances also stimulated in typical biological matrices (e.g. trypto phan u. a.), so that their fluorescence does not differ from that of the marker mole küls can be distinguished, which extremely ver the detection limit worsened. Only the marking with special fluorescence zenzmarker using the method of time-delayed fluorescence can work around these problems. However, this method requires more expensive devices and is practically only by means of so-called enhancement steps done to increase sensitivity, but what means an additional operation.

In German patent application DE 39 16 432 A1 from 1990 a potentiometric method for the detection of an immuno-reaction between unmarked complementary partners described which but also for smaller analyte molecules in one area below 1 ppm is applicable.

For immunoassays that operate like affinity chromatography and in which a displacement reaction with a marker The state of the tech describes the analyte molecule Up to now, only measurements using flow measuring cells in which the Interaction time (= pure measuring time) with the measuring quantities (Light, electricity etc.), given by the flow velocity ben, is relatively short (only a few seconds). Such a short one Measuring time does not allow high elec. Especially with flow measurements tronic damping. Hence the electronic or optoelek tronic amplification technology through the most sensitive Areas of strongly falling signal-to-noise ratio (S / R-Ver ratio) set limits. Are also impossible with the fast len flow of the labeled substance through the flow De Repeat measurements that are similar when averaging positive effect on the S / R ratio. Furthermore repeat measurements need a reference point, d. H. a sub basic measurement (blank value measurement or blanc value), the one appropriate device requires.  

With a simple and quasi-automatic separation of the sample matrix emerging from the immunological reaction zone with disruptive components except in connection with the heterogeneous ELISA techniques that require an additional separation prescribe step, nothing reported in the state of the art. But this is for correct measurements of central importance and an essential requirement for repetitive measurements, which of course involve incorrect measurements also add.

Equalization of immunoreactions with receptor reactions or DNA pairing and molecule-recognizing guest / host molecule Storage with regard to the generally applicable arrangement that can be used by sensors has not yet been described, since labeling with a voluminous enzyme makes these selective Binding reactions very severely hampered (steric disability). This is the case with the relatively small redox systems or IR fluores decorative molecules are not the case.

There is therefore a need for a generally applicable res method and a device that the known Disadvantages of the direct display and also those marked with immu processes running through the biological partners (all current Immunoassays).

The object of the invention to increase the sensitivity while reducing the known cross interference of all traditional immunoassays and similar methods, is achieved in particular through the selected combination of several process steps according to Claim 1.  

To achieve the required extremely low detection limit in the spu Renal analysis area according to the invention instead of the vulnerable and un exact enzymatic signal amplification methods repeatable electro chemical and optical measuring methods together with a special elec tronic signal processing (signal averaging) and ent speaking measuring arrangements applied.

The immuno-partner to be determined (antigen or antibody, mole cool or associated receptor, guest molecule or associated Host molecule, DNA strand and associated complementary sequence) is in the method according to the invention either with a stabi len redox system or a stable, in the infrared range (<700 nm) fluorescent compound durable (e.g. covalently bound) connected. These two marker molecule types were selected after extensive preliminary tests and based on certain criteria, which are hereby disclosed. Are suitable as redox systems preferably inorganic or organic systems with high standards exchange current densities on inert metal electrodes. With the Fluo Resence markers are molecules that are used with inexpensive laser diodes Fluorescence in the far infrared (<800 nm) can be excited, the most advantageous marker molecules.

The method according to the invention is simpler more sensitive, faster, more correct and therefore ultimately also cheaper than all previously described immunoassays. Before direct, extremely sensitive and fast is also more partial Detection of smaller antigens according to the above Disclosures with the procedures described there only one is liquid or not possible at all. Exists here but a large area of application for the new molecular analysis (pollutant analytics) especially in the area of environmental protection or monitoring of maximum workplace concentrations and clinical chemistry (medical diagnosis, pathology, forensic medicine).  

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

In an advantageous version, the labeled analyte molecule previously tied to the immobilized partner. The unmarked Analyte molecule in the sample displaces on contact with the well those, marked partners its marked counterpart (redox or fluorescence-labeled antigen or antibody molecule = labeled Analyte molecule) from the surface bond, which is then replaced by a Liquid flow is transported away.

In another version, the labeled analyte mole becomes the sample added cool in a known ratio and the Mix with the immobilized immuno partners (including all complementary systems mentioned above are to be understood) in Wech interaction. Depending on the concentration of the analyte  competitive different amounts of the labeled analyte molecule bound, with an inverse relation. In both ver sions are the unbound or released marked Analyte molecules in the smallest space again by means of the relevant one immunological (complementary) partner who immobilizes or localized and fixed, collected, concentrated and cleaned by rinsing of adhering sample matrix.

In the device according to the invention, in which an electrochemical and optically accessible surface is present, but also the part Be measured that does not correspond to the eluate collection. At the competitive test in which the surface of the device before Kon clocked with the sample covered with immobilized partner molecules is saturated with its labeled complementary molecule the decrease in the intensity of the marking can also be measured which is proportional to the amount of analyte. The other com petitive test in which labeled and unlabeled analyte molecules together to form the immobilized complementary molecules that are free Binding sites have to be added, the increase in mar cated molecules in this surface area of concentration of the analyte is inversely proportional. Here is the amount of mar labeled molecules that pass through the immunogenic zone unbound, directly proportional to the amount of analyte. Accessible during measurement Surfaces can be seen in such an immunological-ana lytic device the analysis result double (redundant) receive. This twofold determination possibility is new and ver significantly improves the reliability of the entire immunoassay. Errors and malfunctions can result from the lack of agreement of these two measured values can be recognized immediately.

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 trap" zone in a displacement assay (see FIG. 2). The same applies analogously to the method in which the labeled analyte is mixed in a known ratio before each analysis of the sample. By collecting the non-bound labeled and unlabeled analyte molecules in a special capture zone, the concentration of the labeled analyte molecules that can be measured there is directly indicative of 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 the non-enzymatic basis of the invention) is the separation of the specifically bound matrix substances before the actual measurement and the collection or enrichment of the labeled molecules on a fla surface on which unlabelled partner molecules with free Binding sites are firmly attached so that they are the marked Partners who unbound the other immunological binding area have passed through or there through the unlabelled analyte released (dislodged from the binding), specifically bind the. The marked Ana tied up on a flat surface lytmolecules, the amount of which, after calibration, provides information about the (unlabelled) analyte molecules present in the sample allowed, can then, depending on the type of marking, electrochemically or be determined optically, with both long measuring times with high damping as well as cyclical or repetitive measurements with electronic (or computer-based) averaging possible are. According to the laws of statistics, N measurements can be used improve the signal-to-noise ratio by the factor root-N.

Instead of the specifically binding immuno-partner molecules with free binding sites as selective "molecule scavengers" according to the pri Competitive immuno-response may also include other collection phases be used. One is sufficient for lipophilic analyte molecules Zone to be flowed through with oil or wax (or PVC plus Plasticizer or the like) is soaked. Alternatively, you can a material that is used in the reversed phase Chromato is common (e.g. RP-18 stationary phase). The latter lends itself particularly well when ionic analyte moles are present cooler should be held. Here you enter the flow solution appropriately charged, lipophilic counterion. That’s what happens to an ion pair bond in oil or fat, which complicates the passage of the labeled analyte molecules through this molecule catcher zone causes. You will find yourself pouring Enrich and can add carrier solution towards the end of the lipophilic zone  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) as well as thin layer chromatography material supplier can be used be (see Fig. 2). The production of Vorrich lines according to the invention is very simple and efficient. In the area of environmental analytical application of the method disclosed here, the corresponding poly- or monoclonal antibodies or F _ab 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) applied over 75% of the test strip area. After these analyte-recognizing macromolecules have been fixed on the test strip surface, the initial area is immersed in a solution which 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 that are 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 later measurement. At the end of this collection zone, a thin zone with dialysis properties can advantageously be used.

This ensures that the flowing base electrolyte (by the type of Antibody molecules are determined) while this zone can pass through the larger ones but labeled analyte molecules are retained and thereby in a extremely small zone can also be enriched. This arrangement can also in clear glass tubes or cheaper plastic tubes, filled with the relevant chromatographic material be performed. If this tube with the dialysis membrane abge closed, you can do without other collection zones. Here only the unobstructed access of the optical beam paths is required guaranteed, which are preferably not to the side of the tube, but be attached axially. With redox-labeled analyte molecules  len this dialysis film is removably attached so that the inside with the collected, labeled analyte molecules len, which is analytically proportional released in the primary immunozone have been accessible to the planar electrochemical cell be made. Of course, the latter can also be done beforehand microelectronic method firmly on the inside of the dialyzer film applied.

The electrode areas 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 covers a larger concentration range of the analyte, ie the calibration curve changes later than in the planar arrangements into a saturation range. 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 process (addition of labeled analyte molecules at the beginning of the assay), a free end zone of the arrangements explained above can also be used, but in which the solvent evaporates. The selected, stable redox marker molecules as well as 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, and dosing after the primary immunogenic zone is also possible. It is important that the evaporation to concentrate the marker molecules is limited to a small zone (area). Because this means that the solvent transports the molecules to be detected there before it evaporates and leaves its transport cargo behind. Fig. 1 to 2 illustrate the various arrangement options using sketches.

The electrochemical measuring method optimized according to the invention represents a cyclic voltammetry in connection with a stable redox system represents, the latter of all possible records that it has a particularly high standard exchange current density owns. Cyclic voltammetry guarantees thin-film measurement cells that have no electrochemical measuring substance implemented, d. H. is consumed. This is always only oxidized  and reduced. The highest sensitivity is fiction according to achieved when the potential range in which the work electrode (s) is operated cyclically, the half-stage potential of the Includes redox markers (+/- 200-600 mV). With high potential Change speeds (»500 mV / sec scan rate) arise at Working electrodes <10 µm anodic and cathodic current peaks, which are proportional to the concentration of the redox system. The higher the scan rate the bigger the current peaks become what a Increase in sensitivity equals. Are used as redox markers, Systems with the highest standard exchange current densities (e.g. Ferrocene compounds, ruthenium complexes, hexacyanoferrate II / III, J⁻ / J u. a. used, then do not interfere completely separated Redox system from the sample matrix much less if with short zen cycle times (faster voltage ramps). The the best, reversible redox systems leave well over 1000 cyclic voltammograms per second (1000 Hz!). Remainders of less reversible, possibly disruptive 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 resulting in no consumption or net sales of the responsive system lead, d. H. also the method of pulse voltammetry or diffe profitable pulse voltammetry or square-wave voltammetry suitable for this, but cannot be used for fundamental reasons operate cyclically so quickly.

Another method step according to the invention, the advantage liable to the desired extreme sensitivity leads to a special compensation method for scanning at high working electrode potential increasing capacitive current that forms the signal background. A computer-controlled evaluation system is used for this, the works like a scan recorder using a fast AD converter. The compensation of the non-Faraday underground current, the also a crucial one to increase sensitivity Contribution can be done in two ways, by itself or can be used together. With working electrodes with The channel assignment of the scan recorder becomes more than 10 µm in diameter when the potential reversal point is reached also reversed as  whether with an X-Y recorder the voltage on the working electrode recorded against the flowing current (C-V diagram) would. Since this is equivalent to a simple signal addition, compensate the same size per measurement channel (time window or potential) anodic and cathodic currents. Only for those around 60 mV This is not the case for current peaks that are apart.

Another step, which is of great advantage for the aim of this invention, is the electronic averaging, which is only possible through rapid repeat measurements without consumption of the measured substance. With a signal averager, the signal-to-noise ratio (S / R) can also be increased drastically because several thousand cycles can be averaged. Since one can easily scan well over 100 potential cycles per second (»100 Hz) in reversible redox systems, the signal-to-noise ratio can be improved by a factor of 10 in just one second Immunoassay methods with enzyme markers correspond, there are improvements in this S / R ratio which determines the detection limit by several orders of magnitude, without the disadvantages of the sensitive protein molecules (enzymes) interfering here. The second method for suppressing the disruptive capacitive current background in cyclic voltammetry uses an ultramicroelectrode array with individual electrode diameters of less than 10 μm 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). For this purpose, the device according to the invention uses a planar array arrangement of approximately 2000 individual electrodes with a diameter of 3 μm, in which the reference and counter electrodes are already integrated into the surface in an optimized manner. Only one of many possible geometrical arrangements has proven suitable here (see Fig. 3).

The new application of cyclic voltammetry for the development of extreme measuring sensitivities in redox systems or marker molecules with redox groups is possible because no alternation of the medium occurs due to the alternating oxidation and reduction. 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) as well as the counter and reference electrode 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 µA corresponds to a converted amount of substance of approx. 10 ^-10 mol / sec. At the moment, good electrochemical measuring cells also allow reliable current measurements in the nA range, which corresponds to a conversion rate that is a thousand times smaller. Due to the signal averaging, pico and femto ampere currents can now be measured, which leads to conversion rates of 10 ^{-16 and} . 10 ^-19 mol / sec leads.

The desired sensitivity level is obtained with optical detection combination of several, at least two Procedural measures.

The first measure is to use marker molecules that are strongly absorbed in the IR beers, and fluoresce in the far infrared because of that the disruptive fluorescent background of some protein molecules (e.g. with biological matrices) does not occur and which is inexpensive ten laser diodes as excitation sources with high spectral luminance are particularly advantageous. The emitted laser light allows due to its low beam convergence, particularly precise demarcations of the measuring window. Fluorescence markers are used as a further measure molecules whose fluorescence decays particularly slowly, so that one this fluorescence well from the quickly fading of the distracting Can separate molecules in the sample matrix. In contrast to one commercially available arrangement where simply the excitation light is prevented by a shutter, and the actual Measurement of the fluorescent light only after a few milliseconds (after the interfering fluorescence radiation has completely decayed) begins and the evaluation takes place over a certain period of time in the method according to the invention via a direct and faster measuring electronic consideration of the slower Cooldown. For this a lock-in amplifier is used, the one  frequency and phase selective amplification enabled. The sub suppressing the disturbing fluorescence with the fast decay time happens here by a suitable choice of the phase angle on the measurement device. Advantage of this method compared to the count rate method (Photon count) of the commercial version is the higher one Sensitivity because the frequency selective gain only one Fraction of the mostly white electronic noise of the photo receiver gers amplified and because at higher light chopper frequencies Can work what the display speed is drastic increased, so that flow measurements, as usual with the FIA, can still be carried out accordingly sensitively.

This u. a. Advantages are obtained with the invention:

• A) a kind of affinity chromatographic column or one open or accessible to optical and electrochemical measurements Chen surface of any carrier material with immobilized th partner molecules to the molecule to be determined (analyte);
• B) a similar "collecting surface" accessible to the measurement for released labeled analyte molecules (in the event of displacement reaction on the above column or surface, if possible completely with labeled immunogen-bound analyte molecules is documented by unlabelled analyte molecules);
• C) a flat electrochemical measuring cell according to the invention in a potentiostatic consisting of two or three electrodes Circuit with one or more working electrodes, the one Cyclic voltammetry in the electroactive area of the concerned Redox systems (repetitive oxidation and reduction) and those in direct contact with the "collection surface" or the can be brought under A) mentioned;
• D) a fluorescence arrangement using fluorescent laser diodes excitation at emission wavelengths <700 nm and that quickly between an unmarked surface and the places where the optically marked analyte molecules on the surfaces concerned are held oscillating back and forth (alternatively, too Fluorescent markers are used, the time-delayed method  enabled fluorescence, but here the direct and rapid measurement using an electronic phase selective Amplifier is done, which is also done with the help of light guides quickly between an unmarked and marked surface and can oscillate here).
• E) an electronic signal averager or a com computer-aided addition of signal-time curves (time proportio for voltage in electrochemical detection or Measurement location for the optical method). A statistical is averaged electronic noise in the highest sensitivity ranges range to zero.
• F) If both the change in the mar cated analyte molecules in the primary immunogenic zone as well which is recorded in the collection zone, you can next to a double measurement a kind of internal standardization by forming quotients carry out. This improves the reliability of the measurements similar to the internal standardization of emission spec troscopic methods.

The surface with the immobilized immunological partner molecule to 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 be accomplished, among other things, with the help of a suitably chosen electric 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 measuring sensitivity and a large measuring range. Alternatively, the immobilized partner molecules can also be bound to carrier surfaces (e.g. glass beads, chromatographic stationary phases, micro titer plates, etc.) by means of the known techniques using spacer molecules which bind F _c part (in the immobilization of antibodies customary in environmental analysis) .

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. Chroma-tographic thin-film supports are also ideally suited for this, since they are easy to modify on the surface. A preferred type in this context are thin-layer plates with or without basic fluorescence marking, which have already been modified in a so-called reversed phase (RP material). Here, the directed immobilization of the partner molecule type (ie the partner of the complementary molecules that does not represent the analyte, which mutually binds to one another) is particularly successful if one specifically targets one or more long-chain aliphatic residues (C ₆ -C ₂₅ ) towards the epitope region. synthesized or, in the case of antibodies, the genetically engineered F _ab fragments with the specific binding sites can produce 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, in the case of denaturation or deactivation, they can be eluted 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 dynamic as part of a flow injection analysis are automated, d. H. on the unloaded RP waiter The molecules to be immobilized (without bound analyte molecule with marker but also given before) after a rinse to remove the excess in the case of unloaded molecules the labeled analyte molecules until they are sown be passed over the surface. After another The sample can then be rinsed. The use of accessible flat surfaces for the immobilization of the part nermolecules can save one process step, because you can the amount of labeled analyte molecules bound there in both Versions (pre-immobilization with labeled analyte molecules and Competitive loyalty from the one marked with the sample Analyte molecules) both with the planar electrochemical measurement  cell (by simply pressing it onto the surface) as well as with the optical fluorescence method easily determine without one to use additional collection surface.

With both detection methods it is important that interfering substances washed away from the sample matrix before the actual measurements will. This is done by pure, buffered electrolyte solution, which supports both the stability of the biomolecules and the Basic electrolyte for electrochemical or optical detection represents kind.

In the invention, the redox systems selected are those the working electrode material used (platinum, gold, other precious metal) the highest standard exchange current densities demonstrate. This includes preference of all possible redox systems only the particularly reversible systems, such as those based on of ferrocene, ruthenium complexes, hexacyanoferrate (II / III), Iodine / iodide etc. Particularly advantageous redox systems have one Redox potential close to that of oxygen so that it not be disturbed by the latter and in this case the can perform cyclic voltammetry even without inert gas flushing.

The electrochemical cell is particularly advantageous also use a planar microelectrode array arrangement because The advantages of Ultra for single electrode diameters <10 µm microelectrodes occur. The latter are: redox levels instead of peaks in the voltammogram, reduction of the ratio of capacitive to Faraday current, agitation independence, quasi-consumptionless Measurement without cycling the voltage u. a.

As a particularly advantageous class of molecules for fluorescence labeling z. B. bis-isothiocyanate derivative of 2- (4'-chloro-7 '(3''-ethyl-2''- benzothiazolinylidene) -3', 5 '- (1''' - per panediyl) -1 ', 3', 5'-heptatrien-1'-yl) -3-ethylbenzothiazolium bromide on 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 diodes with corresponding IR-sensitive semiconductor detectors enables particularly small measurement setups (e.g. hand-held measuring devices).

The devices and methods of operation according to the invention explained in more detail using examples:

example 1

Displacement of the labeled analyte molecule from the immunological prep surface area through the unlabeled analyte molecule in the sample:

An advantageous embodiment of the arrangement according to the invention and the method according to the invention is based on a competitive Reaction between labeled and unlabeled analyte molecule. Here compensate for non-specific connections because they are in both Cases occur to the same extent. It is also desirable that the unlabeled analyte molecule has a slightly lower binding has constant than the marked, to more effectively displace it to be able to. This is usually the case because the covalent ver linkage with a relatively large marker molecule the antibody Antigen (or generally: molecule complement molecule) binding weakens because both molecules can no longer optimally approach each other nen.

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 bound to the surface of Sepharose material with a small grain size (150 µm) using known immobilization techniques and coated with fluorescence-labeled HSA. A europium chelate (BCPDA) was chosen as the fluorescence molecule. Several fluorescent chelate complexes could be covalently attached to one HSA macromolecule (20-30). In this example, the europium chelate compound 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 unmarked HSA was added, the HSA molecule provided with the fluorescence marker and displaced from the surface of the Sepharose particles loaded with HSA antibodies was measured 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 at the lock-in amplifier. Fig. 5 shows the signals that can be measured by adding unlabelled HSA. They are proportional to the HSA concentration of the sample placed on the column. With this measuring technique, significantly more samples can be analyzed per unit of time than with the photo 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 ferrocene molecule in 6 steps using standard methods of organic synthesis. The selected, very reversible redox system had to be carried out beforehand in a water-soluble form by adding strongly polar side 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 cleaning 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-labeled atrazine in extremely high dilution on a carrier surface. Due to the direct contact of the redox system with the working electrode surface and the absence of diffusing or diffusing redox moles, the cyclic voltammogram results in sharper peaks than usual. This effect also increases sensitivity.

Example 3

Repetitive electrochemical detection limit increased 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 voltammetry is in the millimolar concentration range. The method of electronic signal averaging, which can be used here due to the consumption-free measuring technology, can, as Fig. 8 shows, still extremely low redox concentrations far below the micromolar (µmol / 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 agent concentration (compared to the usual 0.1 Hz mode of operation) or a correspondingly increased electron transfer number corresponds to what would be of equal importance with 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 the case of fast cycles voltammograms by applying the cyclic mean education:  

As is well known, a so-called capacitive current occurs at fast potential change speeds because of 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 cyclic voltammetry because this Share far exceeds that of the relevant redox peak (Faraday current) and leads to voltammograms, as shown in Fig. 9. If, on the other hand, the voltage coordinate values for macro working electrodes are counted down to the individual digi tal channels of the averaging device or scan recorder using this device, similar to an XY recorder, then the reversal of the sign of the current during addition leads to one and the same Channel (which 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 capacity (with the same working electrode potential is proportional except for 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 by this circuit trick. Since they run at different potentials, a low-noise, averaged cyclic voltammogram is obtained, which looks as shown in Fig. 8. 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 a Ultra Micro Electrode Arrys:

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 possible. In addition, the changed diffusion conditions for the electroactive species to a different signal form. In contrast to planar diffusion in macroelectrodes, ultra-microelectrodes (diameter <10 µm) allow spherical diffusion conditions and convert so little substance that there is no depletion layer growing into the solution, which is indicated in the voltammogram by a clear step and a stable one Current limit plateau shows. Fig. 10 shows the difference between a cyclic voltammogram of a trace of a redox system in a macroelectrode and an ultra-microelectrode array of the geometrical arrangement shown 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. Next the principle of paper and thin layer chromatography can also other carriers are used for the immobilized partner molecules will. An example of this can be a piece of a columnar Make chalk (or sintered glass / ceramic). Here at hapten-like analytes the antibody molecules before Immobilization on the chalk surface with the labeled analyte saturated molecules. Immobilization can be done by simple Immerse yourself in this solution, taking the running route can watch. The molecular associations are determined using bifunctional cross-linked reagents or fixed to the surface. From this primary immunological zone is separated by a untreated flow section, establish a second molecule collection zone tet. For reasons of high sensitivity, this is sufficient narrow area directly at the end of the chalk support. In the case the end surface is provided with the same antibodies, which were also used in the primary immunological zone  only with the difference that here the analyte-selective binding are free, so that they are the by the unlabelled analyte Labeled analyte molecules released in the sample are released again Gather and concentrate in the smallest space. The electrochemical or fluorometric measurement then takes place on the polished end surface of the chalk column. The flow of a carrier electrolyte or the sample by the capillary forces by placing a maintain absorbent graft on this end face. The measured sample quantity must be taken from the decrease in the sample volume be determined.

In the invention, a carrier (z. B. flow column, flow paper, membrane filter, etc. to which a partner is bound is done by means of a competitive reaction displaced the unlabeled analyte. However, he will be at the exit of the Before the measurement again, the immobilized complement column molecules bound and thus prepared for the actual measurement chert, while also annoying accompanying substances by rinsing can be removed before the extremely sensitive electrochemi cal or optical measurement is carried out repetitively and by The signal-to-noise ratio is arbitrary is increased.

Literature:

1) S. Wüst, U. Doht, Th. Giersch, Ch. Wittmann, B. Hock, GIT, Fachz. Lab. 2 (1990) 99-106.
2) B. Hock, Z. Wasser-Abwasser-Forsch. 22: 78-84 (1989).

Claims (28)

1. A method for carrying out particularly sensitive immunoassays and other assays which are based on molecular receptor, DNA complementary DNA strand and guest-host molecule interaction forces, characterized by 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. 2. Device according to claim 1, characterized, that as analyte-recognizing molecules or molecular associations the respective partner of the extremely selectively connecting Molecule pairs: Antibody (antibody fragment with binding site) - anti gene, molecule - receptor, DNA segment - more complementary DNA section and guest molecule - host molecule on special waiter areas and used immobilized in certain zones will. 3. Device according to claim 2, characterized, that the partner molecule complementary to the analyte at a statio när carrier is immobilized in two separate zones, wherein the molecules of the first zone at the beginning of an analysis with the redox- or fluorescent-labeled analyte molecule the, so that thereby all binding sites are blocked and at Contact with the unlabelled analyte molecule from the sample Displacement of the labeled analyte molecules takes place, the latter tere but elsewhere in the device on the smallest possible Volume can be collected again before being electrochemical or optically repetitive up to the desired signal / noise ratio will measure. 4. Apparatus according to claim 2 and 3, characterized, that besides the displacement reaction also a competitive binding reaction between the unlabeled analyte molecule in a sample and the redox or fluorescent labeled analyte molecule that everyone Sample in the known ratio must be added to the carrier bound partner molecules can run off, here after rinsing  of the unbound molecules and removal of the sample matrix repetitive electrochemical or optical measurement can take place. 5. The device according to claims 2-4, characterized, that the relevant analyte-recognizing complement molecules on Trä surfaces are immobilized that the repetitive electro enable chemical or optical measurement, i.e. either intimate contact with a planar electrochemical three-electro allow the measuring cell or the measurement of the IR fluorescent light enable. 6. The device according to claim 2-5, characterized, that as a carrier for the analyte fixed at certain points recognizing and selectively binding complement molecules next to the typical microtiter material polystyrene preferably chromato graphic stationary phases, such as adsorbent-filled columns, RP-18-like materials, flow paper strips, membrane filter strips cellulose-based or the like, which have capillary flow properties, be used. 7. The device according to claim 2-6, characterized, that on a sample run (kind of affinity chromatographic Miniature column) at least one primary immunological (or gen my: analyte-specific binding) zone if necessary together with another collection and enrichment zone is available.   8. The device according to claim 7, characterized, that the affinity chromatographic separation, displacement or competitive connection is carried out either planar, so that the planar three-electrode electrochemical electrode is unimpeded Find contact with different surface sections can and the optical measurement alternatively between a Trä background and the measuring zones can oscillate. 9. The device according to claim 7, characterized, that as a collection and enrichment phase, narrowly defined zones in the Sample flow direction behind the primary immunogenic zone (above Definition) created from the relevant analyt recognizing and binding complement molecules exist, but have free binding sites and the redox-active thus collected or fluorescent analyte molecules of repetitive measurement respectively. 10. The device according to claim 9, characterized, that as the second collection and enrichment zone the lipophilicity of the labeled analyte molecules is used in that instead of the complement molecules in question a lipophilic zone with oil or wax-soaked surface or RP-18 material used is, with ionic analytes of this phase still a lipophilic Counter ion is added to the ion pair formation.   11. The device according to claim 9, characterized, that the collection and enrichment phase for those in the primary Labeled analyte molecules displaced by a reaction zone selected according to the molar mass of the labeled analyte molecule Dialysis membrane is given, which is at a certain distance from the pri reaction zone is attached, and both stationary with evaporated electrodes and disassembled electrochemically can be measured. 12. The device according to claim 11, characterized, that this dialysis membrane is also attached to the flow direction first surface is the analyte-binding complemen contains immobilized molecules. 13. The apparatus according to claim 9, characterized, that the collection and enrichment zone is achieved in that the solvent by applying heat and transport gas on Is evaporated at the end of the sample run. 14. The device according to claim 2-13, characterized, that the primary analyzer-recognizing and binding reaction zone and the separate collection and enrichment zone within one test strip-like carrier with capillary forces, so that the transport of the liquid sample through the different zones  can be achieved by the latter and no external pump is necessary is agile, with the evaluation over concentration-proportional distances or integral signals over the characteristic route sections done and is also redundant. The evaluation device is a scanner with optical or electrical measurement. With electrochemical scanning roller-shaped working electrodes used. 15. The apparatus according to claim 2-14, characterized, that the measurement of the concentration of the redox-labeled analyte moles cool on the corresponding surface zones of the invention Device by the electrochemical method of fast cyclic voltammetry or another method without Net sales (substance consumption) works, using a planar Three-electrode measuring cell, together with a suitable reason electrolyte is pressed onto these points, takes place (principle of Thin-film measuring cell). 16. The apparatus of claim 15, characterized, that the planar or quasi-planar electrochemical three-electro denmeßzelle instead of a macro working electrode made of a noble metal, such as platinum, gold or the like, from an ultra-microelectrode array the same materials with single electrode diameters <10 µm contains to the strong in fast cyclic voltammograms to decrease the increasing capacitive current. 17. The apparatus of claim 15 and 16, characterized, that the working electrode or the ultra-micro used for this electrode array integrated into the relevant zones of the assay are.   18. The apparatus according to claim 15-17, characterized, that the cyclic voltammogram with a high repetition rate in Range 1 to »100 Hz not as usual on an X-Y recorder, but regi on an electronic averager striert, which is switched so that the digitized Current signals in both potential directions on one and the same voltage-proportional channel are added, which is because of the under different sign of the capacitive current share in the different directions of change of an almost 100% com compensation of this disturbing 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, that the ultra-microelectrode array is used as the working electrode becomes. 20. The apparatus of claim 15-19, characterized, that the measuring process of an analyte is automatically computer controlled in follows the way that both the picked up by the arrangement Sample quantity and the collection time reproducibly observed the and the repetitive electrochemical measurement with each same number of addition cycles by the Signal / noise ratio depends, is carried out.   21. The apparatus according to claim 20, characterized, that as redox marker molecules that are covalent to the analyte molecule linked, redox systems with a particularly high standard exchange current density can be used, which is in the concerned Dissolve solvent or in the sample matrix. 22. The apparatus according to claim 21, characterized, that as particularly reversible redox systems, water-soluble, stable and storable ferrocene derivatives, ruthenium complexes, hydroquinones, Hexacyanoferrate (II / III), iodine / iodide or the like can be used are connected to the analyte molecule via spacer molecules. 23. The device according to claims 2-14, characterized, that as marker molecules for repetitive optical detection sta bile, storable special molecules are used that light absorb the wavelength <700 nm and in the longer wavelength range emit again as fluorescent light. 24. The device according to claim 23, characterized, that as a class of molecules for advantageous IR fluorescence labeling the bis-isothiocyanate derivative of 2- (4′-chloro-7 ′ (3 ′ ′ - ethyl-2 ′ ′ - benzothiazolinylidene) -3 ′, 5 ′ - (1 ′ ′ ′ - propanedyl) -1 ′, 3 ′, 5′-hep- tatrien-1'yl) -3-ethylbenzothiazolium bromide, porphyrin derivatives with large 2n + 1 Pi electron systems or phthalocyanines  be used, which are also characterized by particularly high extinction mark coefficients. 25. The device according to claim 2-14, characterized, that the repetitive optical measurement that the averager is achieved by alternating the to mes send in the fluorescent surface zone and the substrate one not with the collection phase or the complementary molecules covered area is measured, which is indicated by a relative movement of the optical beam can be achieved. 26. The device according to claim 2-14, characterized, that as an optical excitation source instead of a white light source and a monochromator an intermittently operated laser diode in the IR wavelength range with high radiation density, mono chromasie and no stray light together with a lock-in ver strengthening technology is used. 27. Procedure for determining a partner's concentration complementary pairs of molecules that recognize each other chemically selectively (Antibody antigen, receptor receptor molecule, DNA sequence comple mental DNA strand, guest-host molecule) by means of a Immunoassays according to claims 1-26, characterized, that redox and fluorescence-labeled analyte molecules are used the and the electrochemical or optical measurement of the distribution of these labeled molecules in the different zones of the test direction without material consumption, so that any number  repetitive measurements are possible. 28. The method according to claim 27 and claim 1, characterized, that the repetitive measurements in a reproducible manner be added to a signal mean, so that the Signal / noise ratio between the Faraday current and the Background as well as the surface with the fluorescent molecules and the free carrier surface is significantly improved.