DE4216696C2 - Method and apparatus for increasing sensitivity and selectivity in immunoassays, molecule receptor, DNA complementary DNA and guest-host molecule interaction assays - Google Patents

Description

The invention is directed to a method for sensitivity and Selectivity increase in immunoassays, molecule receptor, DNA com complementary DNA and guest host molecule interaction assays according to claim 1 and a device for carrying it out according to claim 17.

The quantitative analysis of complex substance mixtures using the very selective antibody-antigen binding (key-lock principle) and the DNA pairing in the so-called DNA probes is in the bio chemistry and clinical chemistry an established analytical method and, demented widespread speaking. Competitive tests are usually marked with Antigens, antibodies or DNA molecules are used. At the radio immuno ace say (RIA) is the antigen (or antibody) added to the sample molecule in the sandwich method) radioactively labeled. With enzyme immu no-assay (EIA or the heterogeneous enzyme-linked adsorbend assay, ELISA) is a so-called marker enzyme on the relevant marker molecules bound. These labeled molecules compete with the un measured labeled molecules (substance to be determined = analyte) to bind 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 comparison, a test with an antigen (DNA molecule) of known concentrations tration is carried out. The measurement signals are reversed with this method returns proportional to concentration. In the so-called sandwich tests labeled antibodies used. These bind to antigen moles in a sandwich cool, which in turn depends on the concentration on carrier-bound Antibodies are bound.  

The very specific binding of two complementary molecules allows na vice versa, of course, the quantitative determination of the larger partner (Antibody, receptor analysis or the complementary DNA molecule (or organic surface), which has a com has complementary DNA sequence to the labeled sequence) and are therefore the most important biochemical analysis methods. After it meanwhile is possible, even for smaller molecules (haptens) monoclonal antibodies To produce in any quantity, these are immunological metho which is also increasingly important for environmental analysis. For example using monoclonal antibodies for the various di oxine significantly reduce the total analysis costs by only using positive result of the immunological test the very expensive GC-MS Ana lysis must be carried out (screening).

The now traditional immunological determination methods (RIA and EIA or ELISA) have considerable disadvantages that are sufficient are known. Either occur because of the use of radioactive material Disposal, shipping and storage problems or you have to with a Be influence of the immunological reaction by the most, 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 The lifespan of the enzyme is limited and the enzymatic reaction (Bio catalysis) in the case of unknown environmental matrices by inhibitors or En zympoisons (e.g. heavy metals) are disturbed, so that wrong Results occur. Usually the enzyme-labeled compound from Stabi be stored in a cool place for reasons of quality. Because of this diminishing activity additional calibration steps are required and the GLP guidelines (Good laboratory practice), which are followed in quantitative analyzes need a lot of the more expensive enzyme through reserve samples marked connections. Phase separation is also required, a Substrate has to be added, which makes handling very complicated becomes. The required work steps usually require some Hours. The so-called homogeneous enzyme immuno-As run without phase separation  says (EMIT) who work competitively and are only for small antigen molecules are suitable.

The known methods are u. a. in S. Wüst, U. Doht, Th. Giersch, Ch. Wittmann, B. Hock, GIT, Fachz. Lab. 2 (1990), pages 99 to 106 [1] and B. Hock, Z. Wasser-Abwasser-Forsch. 22 (1989), 78 to 84 [2].

With a simple and generally applicable analytical-chemical advantage selective molecular-receptor interaction forces, DNA pair image reaction and equally selective guest-host relationships of supramolecular chemistry for the purpose of quantitative determination or only a few exist for the construction of corresponding sensors Preparatory work.

The advantageous structure of a simple and inexpensive electrochemical measuring system, which in the form of potentiometry or amperometry requires the least amount of equipment, usually fails because that the substance to be determined (analyte) is not a potential one Ion, but as a neutral molecule or not electrochemical can be implemented selectively. The use of optical methods (spec tralphotometry, fluorescence measurement, ellipsometry, surface plasmon Re sonance spectroscopy, evanescent wave method, etc.) for acquisition The specific molecular interactions mentioned above are solved been written. Some can do without the immunological response marked partner due to the change in the refractive index of a Detect phase boundary, but have big problems with small analyte moles cool (as usual in environmental analysis) in the trace range (<1 ppm), because the non-specific binding of disruptive accompanying substances, which even usually in a large excess next to the analyte in a real sample are present, is sensitivity-limiting.

Optical methods with the usual fluorescent markers, which an anre wavelength in the UV or visible range require egg high expenditure on equipment, that of a widespread routine application  is in the way. The effort to stabilize the light source Monochromatization using a stray light-free monochromator Avoidance of extraneous light influences, to increase sensitivity etc. requires apparatus structures that are 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 u. a.), so that their fluorescence does not differ from that of the marker mole küls can be differentiated, which the detection limit extremely ver worsened. Only the marking using a special fluorescence marker ker who work according to the method of time-delayed fluorescence can work around these problems. However, this method requires more expensive equipment and is practically only by means of so-called enhancement steps to increase the Sensitivity performed, but what an additional operation means.

In German patent application DE 39 16 432 A1 from 1990 a po tentiometric method for the detection of an immuno-reaction between described unmarked complementary partners, which also can be used for smaller analyte molecules in a range below 1 ppm.

For immunoassays that are operated like affinity chromatography and where a displacement reaction with a labeled analyte The state of the art has so far only described molecule utilization Measurements using flow cells in which the interaction time (= pure measuring time) with the measuring variables (light, electricity etc.), through which Flow rate specified, relatively short (only a few seconds) is. Such a short measuring time allows especially with flow measurement no high electronic damping. Hence the electronic or optoelectronic amplification technology through the in the sens most significant areas, the signal-to-noise ratio drops sharply (S / R ratio) set limits. Also impossible with that rapid flow of the marked substance through the flow De tector Repeat measurements that are similarly posi affect the S / R ratio. They also need repetition measurements a reference point, d. H. a background measurement (blank value measurement solution or engl. blanc value), which requires a corresponding device.  

With a simple and quasi-automatic separation of the also the sample matrix emerging from the immunological reaction zone the components are except in connection with the heterogeneous ELISA techniques that require an additional separation step nothing reported in the prior art. But this is for real measurement solutions of central importance and an essential prerequisite for repe titive measurements, in which incorrect measurements are of course also ad dieren.

Equalization of immunoreactions with receptor reactions or DNA pairing and molecule-recognizing guest / host molecule incorporations with regard to the generally applicable sensory exploitation according to the invention baren arrangement has not been described so far because the marking with a voluminous enzyme, these selective binding reactions are very strong disabled (steric disability). This is with the relatively small Re dox systems or IR fluorescent molecules are not the case.

The invention has for its object a method that the known disadvantages of directly displaying and also the procedure taking place with marked partners avoids, and a device to specify to carry out the procedure.

This object of the invention is through a process solved according to claim 1 or by a device according to claim 17.

To achieve the required extremely low detection limit in the trace According to the invention, the analysis area is replaced by the susceptible and inaccurate exact enzymatic signal amplification methods repeatable electro chemical and optical measuring methods together with a special elec tronic signal processing (signal averaging) and correspond appropriate measuring arrangements applied.  

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 invented method according to the invention either with a stable redox system or egg A stable, fluorescent compound in the infrared range (<700 nm) durable (e.g. covalently bound) linked. Choosing these two Marker molecule types were carried out after extensive preliminary tests and on According to certain criteria, which are hereby disclosed. As a redox sy systems are preferably suitable with inorganic or organic systems high standard exchange current densities on inert metal electrodes. Both Fluorescence markers are molecules that are used with inexpensive laser diodes Fluorescence in the far infrared (<800 nm) can be excited most advantageous marker molecules.

The method according to the invention is simpler, more sensitive, faster, more correct and therefore ultimately cheaper than all previously written immunoassays. The direct, extreme is also more advantageous sensitive and rapid detection of smaller antigens that are after the above-mentioned writings with the Ver driving poorly or not possible at all. Especially 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 the clini chemistry (medical diagnosis, pathology, forensic medicine).

The process according to the invention can be carried out differently according to the examples:

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

In another version, the labeled analyte molecule in the sample added to a known ratio and the mixture is left with the immobilized immuno-partners (including all of the above mentioned systems must be understood) interact. Depending on the Concentration of the analyte will be different amounts of the labeled Analyte molecule competitively bound, with an inverse relation be stands. In both versions, the unbound or released labeled analyte molecules in the smallest space again using the be meeting immunological (complementary) partner who immobilizes or is 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 the part can also be measured that do not correspond to the eluate collection. In the competitive Test in which the surface of the device before contact with the sample is covered with immobilized partner molecules that are labeled with their th complementary molecule are saturated, the decrease in In intensity of the label can be measured, the proportion of the analyte proportion nal is. In the other competitive test, in which marked and unmar analyte molecules together to form the immobilized complementary molecules cool that have free binding sites added is the increase of labeled molecules in this surface area of the concentration tion of the analyte is inversely proportional. Here is the amount of mar labeled molecules that pass through the immunogenic zone unbound Amount of analyte directly proportional. Surface accessible during measurement Chen you can with such an immunological-analytical Vor receive the analysis result twice (redundant). These two Easy determination is new and improves reliability of the entire immunoassay significantly. Errors and faults can be caused by the lack of agreement between these two measured values is immediately recognized the.

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 molecule is mixed 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 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 Ver drive on a non-enzymatic basis) of the invention is the separation of the non-specifically bound matrix substances before the actual one Measurement as well as the collection or enrichment of the labeled molecules a flat surface on which unmarked partner molecules with free Binding sites are firmly connected so that they are the marked partners, that pass through the other immunological binding area unbound or released there by the unlabelled analyte (from the Binding), specifically bind. The one on a flat Surface-bound labeled analyte molecules, the amount according to Ka libration an information about the present in the sample (unmar kiert) analyte molecules allowed, depending on the type of markie tion, electrochemically or optically, both being long Measuring times with high damping as well as cyclical or repetitive measurements possible with electronic (or computer-aided) averaging 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 primary competi Other immunization phases can also be used. In the case of lipophilic analyte molecules, one zone to flow through is sufficient, which is soaked with oil or wax (or PVC plus plasticizer or similar),  out. Alternatively, a material can be used for this, that in reversed phase chromatography is common (e.g. RP-18 stationary Phase). The latter is particularly useful when ionic analyte molecules should be recorded. Here you give the Flow solution to a correspondingly charged, lipophilic counterion. Thereby there is an ion pair bond in the oil or fat, which is an Er Difficulty in the passage of the labeled analyte molecules through this molecule catcher zone causes. You will find yourself with a flowing carrier solution accumulate towards the end of the lipophilic zone and can thus the sensitive Measurement are made accessible.

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 can be used. Be a supplier (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 _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 fixing these analyte-recognizing macromolecules on the surface of the test strip, 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 in order 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 catcher for the later measurement. At the end of this collecting zone, a thin zone with dialysis properties can advantageously be used.

This ensures that the flowing basic electrolyte (by Art of the antibody molecules) can pass through this zone larger labeled analyte molecules are retained and there by also being enriched in an extremely small zone. This to Tidiness can also be found in transparent glass tubes or cheaper pla Microtubes filled with the relevant chromatographic material, be performed. If this tube with the dialysis membrane abge closed, you can also do without other collection zones. Here only the unobstructed access of the optical beam paths must be guaranteed are here, preferably not axially but laterally to the tube to be brought. With redox-labeled analyte molecules, this becomes dialy sier Folie attached removable, so that the inside with the collected, labeled analyte molecules in the primary immunozone have been released in proportion to the analyte, the planar electrochemical cell are made accessible. Of course, the latter can even beforehand by microelectronic methods on the inside of the Dialysis film can be applied.

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 covers a larger concentration range of the analyte, ie the calibration curve passes 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 method (addition of labeled analyte molecules at the beginning of the assay), a free end zone of the above-mentioned arrangements 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 freight there. Fig. 1 to 2 illustrate the various arrangement options using sketches.

The optimized electrochemical method according to the invention is one cyclic voltammetry combined with a stable redox system, the latter being distinguished from all possible in that it ei ne has a particularly high standard exchange current density. The cyclical With thin-film measuring cells, voltammetry guarantees that there are none Measuring substance converted electrochemically, d. H. is consumed. Here is always only oxidized and reduced. The highest sensitivity is achieved according to the invention if the potential range in which the Working 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 with working electronics the <10 µm anodic and cathodic current peaks that the Konzentra tion of the redox system are proportional. The higher the scan rate the more the current peaks become larger, which increases the sensitivity equals. Are used as redox markers, systems with the highest standard exchange current densities (e.g. ferrocene compounds, ruthenium complexes, Hexacyanoferrat II / III, J⁻ / J u. a.) used, then do not interfere fully continuously separated redox systems from the sample matrix much less, if working with short cycle times (fast voltage ramps) becomes. The best, reversible redox systems are definitely not enough 1000 cyclic voltammograms per second (1000 Hz!) Too. Remainders of less reversible, possibly disruptive redox systems from the Pro benmatrix cannot be implemented as quickly electrochemically and he therefore do not produce a measurement signal (current flow).  

Alternatively, other electrochemical methods can be used do not lead to consumption or net sales of the reacting system, d. H. also the method of pulse voltammetry or differential Pulse voltammetry or square-wave voltammetry are suitable for this, can not be cyclically so quickly for fundamental reasons float.

Another method step according to the invention, which advantageously leads to the desired extreme increase in sensitivity lies in egg a special compensation method for those who work quickly electrode potential scan rates increasing capacitive current carrying the signal forms underground. For this purpose, a computer-controlled evaluation system is used turns that ar using a fast AD converter like a scan recorder works. The compensation of the non-Faraday underground current, the also make a decisive contribution to increasing sensitivity can be done in two ways, alone or together can be used. For working electrodes with diameters over 10 µm becomes the channel assignment of the scan recorder when the pot is reached tial reversal point also reversed, as if with an X-Y recorder the voltage at the working electrode against the flowing current C-V Diagram) would be recorded. Since this is a simple signal addition equals, compensate for each measuring channel (time window or potential) equally large anodic and cathodic currents. Only for the approx. This is not the case with current peaks 60 mV apart.

A further step, which is of considerable advantage for the aim of this invention, is the electronic averaging that is only possible through rapid repeat measurements without using the measured substance. With a signal mean value generator, the signal / noise ratio (S / R) can also be increased drastically because several thousand cycles can be averaged. Since it is easy to 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. At measuring times that match those of the immuno -Assay method with enzyme markers, there are improvements in this S / R ratio determining 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 interfering capacitive current background in cyclic voltammetry uses an ultramicroelectrode array as the working electrode with individual electrode diameters of less than 10 μm. There are no longer any anodic and cathodic current peaks, but rather 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 in the surface in an optimized manner. Only one of many possible geometrical arrangements has proven to be suitable here (see Fig. 3).

The new application of cyclic voltammetry to achieve extreme measuring sensitivity 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-layer 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 for 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. 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. The signal averaging now makes it possible to measure pico and femto amper currents, which leads to conversion rates of 10 ^{-16 and} . 10 ^-19 mol / sec leads.

The desired sensitivity is obtained with optical detection increase through the combination of several, at least two, procedures measures. The first measure is to use marker molecules that are in the Strongly absorb IR, and fluoresce in the far infrared because thereby the disruptive fluorescent background of some protein molecules (e.g. with biological matrices) does not occur and the inexpensive La Serdiodes especially as excitation sources of high spectral luminance are advantageous. The emitted laser light allows due to its low Beam convergence particularly precise boundaries of the measurement window. As white tere method one uses fluorescent marker molecules, their fluorescence decays particularly slowly, so that this fluorescence is good from the the rapidly decaying of the interfering molecules in the sample matrix can. In contrast to a commercially available arrangement, at which is simply prevented by a shutter, and the actual measurement of the fluorescent light only after a few milli seconds (after the interfering fluorescence radiation has completely decayed) begins and is integrated over a certain period of time, the evaluation takes place at the inventive method via a direct and faster measuring electronic consideration of the slower cooldown. To do this uses a lock-in amplifier that is frequency and phase selective Enhancement enables. The suppression of disruptive fluorescence The fast cooldown is done here by a suitable choice of Phase angle on the measuring device. Advantage of this method over the counter ten method (photon count) of the commercial version is the higher Sensitivity because the frequency selective gain is only a fraction part of the mostly white electronic noise of the photo receiver ver strengthens and because higher light chopper frequencies are used can, which increases the display speed drastically, so that too Flow measurements, as usual at the FIA, are still very sensitive can be carried out.

The following advantages are obtained with the invention:

• A) a kind of affinity chromatography column or an open one or the optical and electrochemical measurement accessible upper  surface of any carrier material with immobilized partner molecules to the molecule to be determined (analyte);
• B) a similar "collecting surface" accessible to the measurement for released labeled analyte molecules (in case of displacement reaction on the above column or surface as completely as possible is labeled with labeled immunogen-bound analyte molecules, by unlabelled analyte molecules);
• C) a flat electrochemical measuring cell according to the invention in one two or three electrodes existing potentiostatic circuit with one or more working electrodes that form a cyclic voltamm in the electroactive area of the redox system in question (rep titive oxidation and reduction) and the immediate Contact with the "collecting surface" or the one mentioned under A) can be brought;
• D) a fluorescence arrangement, which uses laser diodes for fluorescence at emission wavelengths <700 nm and that quickly between an unmarked surface and the places where the optically mar analyte molecules on the surfaces concerned are oscillated back and forth (alternatively, fluorescence markers are used which are the method of time-delayed Allow fluorescence, but here the direct and fast Measurement using an electronic phase selective amplifier takes place, which also quickly with the help of light guides oscillate back and forth on an unmarked and marked surface can).
• E) an electronic signal averager or a computer support addition of signal-time curves (time proportional to Voltage during electrochemical detection or to the measuring location at optical process). A statistical electro is averaged African noise to zero in the highest sensitivity ranges.  
• F) If both the change of the marked analyte molecules in the primary immunogenic zone as well as in the collection zone is recorded, you can also take a double measurement carry out a kind of internal standardization by forming quotients ren. This improves the reliability of the measurements similar to in 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 be attached to carrier surfaces (e.g. glass beads, chromatographic stationary phases, microtiter plates, etc.) by means of the known techniques using spacer molecules which bind the F _c part (in the case of immobilization of antibodies customary in environmental analysis) become.

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, 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). The directed immobilization of the partner molecule type (ie the partner of these mutually binding complementary molecules that does not represent the analyte) succeeds particularly well here if one targets these molecules with one or more long-chain aliphatic residues (C₆-C₂ gegenüber ) synthesized or, in the case of antibodies, the genetically generated 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, 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-film plates based on an aluminum foil, the latter can also be used elegantly in the electrochemical detection method as a counter electrode.

This process can also be done dynamically as part of a flow injection analysis be automated, d. H. towards the unloaded RP surface next the molecules to be immobilized (without bound analyte molecule with marker, but also with) before removing after rinsing the excess in the case of unloaded molecules the labeled analyte molecules are passed over the surface until saturation. To a new rinse can then be applied to the sample. The Ver use of accessible flat surfaces for the immobilization of the Partner molecules can save one procedural step, because you can do that Amount of labeled analyte molecules bound there in both versions (Pre-immobilization with labeled analyte molecules and competition of the labeled analyte molecules added to the sample) both with the planar electrochemical measuring cell (by simply pressing on the surface) as well as with the optical fluorescence method agree without using an additional collection interface.

With both detection methods it is important that interfering substances are removed the sample matrix before the actual measurements. This is done by pure, buffered electrolyte solution, which both the stabilizer lity of the biomolecules supports the basic electrolyte for the represents electrochemical or optical type of detection.  

The redox systems selected in the invention are those that are the working electrode material used (platinum, gold, other precious metals tall) show the highest standard exchange current densities. Which includes of all possible redox systems, preferably only the particularly rever sensitive systems, such as those based on ferrocene, ruthenium complexes, Hexacyanoferrate (II / III), iodine / iodide etc. Particularly advantageous redoxsy systems have a redox potential close to that of oxygen, so that they are not disturbed by the latter and in this case the can perform cyclic voltammetry even without inert gas flushing.

In the electrochemical cell, egg can also be particularly advantageously ne planar microelectrode array arrangement) because with single electrode diameters <10 µm the advantages of ultramicroelectrodes occur. The latter are: redox levels instead of peaks in the voltammogram, Reducing the ratio of capacitive to Faraday current, Stirring independence, quasi-consumption-free measurement even without the voltage to cyclize u. a.

A particularly advantageous class of molecules for fluorescent labeling are, for. B. bis-isothiocyanate derivative of 2- [4'-chloro-7 '(3''-ethyl-2''- benzothiazolinylidene) -3', 5 '- (1''' - prop-anediyl) - 1 ′, 3 ′, 5′-heptatrien-1′-yl] -3-ethylbenzothiazolium bromide, large porphyry 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 appropriate IR-sensitive semiconductor detectors allows particularly small test setups (e.g. handheld devices).

The devices and methods of operation according to the invention are based on Examples explained in more detail.  

example 1 Displacement of the labeled analyte molecule from the immunologically prepared one Surface area due to the unlabeled analyte molecule in the sample

An advantageous embodiment of the arrangement and of the method according to the invention is based on a competitive reaction between labeled and unlabeled analyte molecule. Compensate here non-specific connections, since they are the same in both cases Extent occur. It is also desirable that the unlabelled analyte molecule has a slightly lower binding constant than the marked one points in order to be able to displace it more effectively. This is usually the case Case because the covalent linkage with a relatively large marker mole cool the antibody-antigen (or in general: molecule complementary molecules) cool) weakening because both molecules no longer optimally sew can hear.

In this example, human serum albumin (HSA) was chosen as the model analyte. The associated anti-body (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 an HSA macromolecule (20-30). In this example, the europium chelate compound was used instead of the IR light-emitting marker because the fluorescence occurring here decays much slower than that of the disruptive 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 unlabelled HSA was added, the HSA molecule displaced with the fluorescent marker 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 on 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 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 appropriate 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 converted into 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. 5.

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-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 molecules, the cyclic voltammogram results in sharper peaks than usual. This effect also increases sensitivity.

Example 3 Repetitive electrochemical oxides increase the detection limit tion 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 classic cyclic voltammetry is in the millimolar concentration range. The method of electronic signal formation, which can be used here due to the consumption-free measurement technology, can, as Fig. 8 shows, extremely low redox concentrations far below the micromolar (µmol / L) range with a very good signal / noise ratio determine. At a cycle frequency of approx. 1000 Hz, for example, 1000 oxidation and reduction reactions can be carried out in one second, which in terms of the electrochemical signal is synonymous with 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 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 the case of fast cyclical ones Voltammograms using cyclic averaging

As is known, at so-called potential change velocities due to the corresponding charge reversal of the double-layer capacitance at the working electrode interface, a so-called capacitive current occurs, the size of which is unfortunately proportional to the rate of change, so that an extremely fast cycle speed should be excluded in the cyclic voltammetry because this proportion far exceeds that of the relevant redox peak (Faraday current) and leads to voltammograms, as shown in Fig. 9. If, on the other hand, if the voltage coordinate values for macro working electrodes are counted down to the individual digital channels of the averaging device or scan recorder with this device, similar to an XY recorder, the reversal of the sign of the current strength during the addition In leads to one and the same channel ( which is assigned a precisely defined working potential) to compensate for 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 rate of change 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. A low-noise, averaged cyclic voltammogram is obtained, which 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 Ul tra microelectrode arrays

If the surface of the working electrode is reduced, the associated double-layer capacity is reduced significantly more than the Fara day current density, i.e. 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 become. In addition, the changed diffusion conditions cause the electroactive species to change their signal form. Ultra-microelectrodes (diameter <10 µm), in contrast to a planar diffusion with macroelectrodes, 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 shows a stable current limit plateau. 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, you can capillary forces are preferably also used. Besides the principle paper and thin layer chromatography can also be used for other supports the immobilized partner molecules are used. An example for can be a piece of columnar chalk (or sintered glass / ceramic) represent. Here the antibody moles are used in hapten-like analytes cool even before immobilization on the chalk surface with the mar saturated analyte molecules. The immobilization can be done by a Multiple immersions are done in this solution, taking the running distance can watch. The molecule associates are bifunctional Reagents cross-linked or fixed on the surface. From this pri The immunological zone is separated by an untreated flow stretch, a second molecule collection zone was established. For the sake of a ho hen sensitivity is sufficient here a narrow area immediately on End of the chalk bearer. In the example, the end surface is the same Chen provided antibodies, which are also in the primary immunological zone were used, with the difference that here the analyte-selective Binding sites are free so that they are the by the unlabelled Analy the labeled analyte molecules released in the sample Gather and concentrate in the smallest space. The electrochemical or fluorimetric measurement then takes place on the polished end face of the Chalk column. The flow of a carrier electrolyte or the sample through  the capillary forces are applied by placing an absorbent plug maintain this end face. The measured sample amount must be from the Decrease in the sample volume can be determined.

If a support (e.g. flow column, blotting paper, Membrane filter etc.) is used, to which a partner is bound this by means of a competitive reaction by the unlabeled analyte displaced. However, it will be at the exit of the column before the measurement again bound by immobolized complement molecules and thereby to actual measurement enriched, while also disturbing loading lubricants can be removed by rinsing before the extremely emp sensitive electrochemical or optical measurement carried out repetitively the signal-to-noise ratio is increased arbitrarily.

Claims (31)

1. A method for increasing sensitivity and selectivity in immunoassays, molecule receptor, DNA complementary DNA and guest-host molecule interaction assays, in which an electrochemical or optical measurement for detecting analytes in samples on the Measurement of accessible surfaces takes place, whereby
an analyte to be determined, in exchange or in competition with a labeled form of the analyte, is bound at least in a first zone on the surface to the complementary partner immobilized there,
the displaced or unbound labeled analyte is collected and enriched in at least one second zone,
the measurement is carried out in both zones or exclusively in the second collecting and enrichment zone, the measurement is carried out repetitively
and wherein a redox or IR fluorescent marker is used for labeling. 2. The method according to claim 1, characterized in that the Analytes complementary partner molecule on a stationary support two separate zones is immobilized, with the molecules of the first Zone at the beginning of an analysis with the redox- or fluorescent-marked Analyte molecule are saturated, so that thereby all binding sites are blocked and in contact with the unlabelled analyte molecule the labeled analyte molecules are displaced from the sample and the latter collected again in the second zone and electrochemically or optically repetitive up to the desired signal / noise ratio will measure.   3. The method according to claim 1 or 2, characterized in that the collection and enrichment in the second zone is achieved by that there the solvent by applying heat and transport gas is evaporated. 4. The method according to any one of claims 1 to 3, characterized in net that each sample redox- or fluorescence-labeled analyte gives and besides the displacement reaction also a competitive binding reaction between the unlabeled analyte in the sample and the right DOX- or fluorescence-labeled analyte on carrier-bound partner molecules le runs off and after rinsing the unbound molecules and Ent distant from the sample matrix the repetitive electrochemical or optical Carries out measurement. 5. The method according to any one of claims 1 to 4, characterized in net that a cyclic voltammogram with a high repetition rate in the range 1 to »100 Hz on an electronic averager is registered, which is switched so that the digitized current signals in both potential directions on one and the same voltage proportional channel can be added. 6. The method according to any one of claims 1 to 5, characterized in net that as a redox marker redox systems with a particularly high standard Exchange current density can be used, which is in the solution in question dissolve medium or in the sample matrix, and the ko with the analyte molecule valent linked. 7. The method according to claim 6, characterized in that as Re dox systems water-soluble, stable and storable ferrocene derivatives, Ruthenium complexes, hydroquinones, hexacyanoferrate (II / III), iodine / iodide are used, which are connected to the analyte molecule via spacer molecules that will. 8. The method according to any one of claims 1 to 4, characterized in net that stabi as marker molecules for repetitive optical measurement  le, storable molecules are used that light the wavelength <700 nm absorb and in the long-wave range again as fluorescence emit light. 9. The method according to claim 8, characterized in that for the IR fluorescence labeling the bis-isothiocyanate derivative of 2- (4′-chlo ro-7 ′ (3 ′ ′ - ethyl-2 ′ ′ - benzothiazolinylidene) -3 ′, 5 ′ - (1 ′ ′ ′ - propanedyl) -1′-, 3 ′, 5′-heptatrien-1′-yl) -3-ethylbenzothiazolium bromide, porphyrin derivatives te with large 2n + 1 Pi electron systems or phthalocyanines become. 10. The method according to any one of claims 1 to 4, 8 and 9, characterized characterized in that the repetitive optical measurement alternately on the fluorescent surface zone to be measured and the support is measured at an uncovered area, which is indicated by a Relative movement of the optical beam is achieved, and that the measuring values are fed to an electronic averager. 11. The method according to any one of claims 1 to 4, 8 and 9, characterized characterized in that a white light source and a monochromator or an intermittently operated laser diode in the IR wavelength range with high radiation density, monochrome and No stray light together with a lock-in amplification technology is used. 12. The method according to any one of the preceding claims, characterized ge indicates that the measurement was averaged becomes. 13. The method according to any one of the preceding claims, characterized ge indicates that when measuring in two zones, a comparison or a Quotient formation takes place. 14. The method according to any one of the preceding claims, characterized ge indicates that for optical or electrochemical measurement Scanner is used.   15. The method according to claim 14, characterized in that for electrochemical measurement roller-shaped working electrodes who used the. 16. The method according to any one of the preceding claims, characterized ge indicates that the measuring process is automatically computer controlled in the Way follows that both the amount of sample taken up by the arrangement as well as the measuring time are reproducibly observed and the repetiti ve electrochemical measurement with the same number of addition cycles is carried out. 17. The apparatus for performing the method according to claim 1, be standing from a carrier,

• immobilized on its surface in at least a first zone analyte-recognizing molecules or molecular associations to which a labeled form of the analyte is bound,
• which has at least one second zone which is capable of collecting and enriching the labeled form of the analyte,
• - And whose surface allows an electrochemical measurement or the measurement of IR fluorescent light in the individual zones.

18. The apparatus according to claim 17, characterized in that as analyte-recognizing molecules or molecular associations the respective Partner of the extremely selectively connecting pairs of molecules: antibodies (Antibody fragment with binding site) - antigen, molecule receptor, DNA section - complementary DNA section as well as guest molecule host ver molecule at the surface in the first and / or the second zone shares are immobilized. 19. The apparatus according to claim 17 or 18, characterized in that the relevant analyte-recognizing complement molecules on carrier surfaces are immobilized, which the intimate contact with a plan allow electrochemical three-electrode measuring cell or the measurement of the IR fluorescent light.   20. Device according to one of claims 17 to 19, characterized records that a sample run with at least a first, ana lysis-specific binding zone together with at least one second, up capture and enrichment zone is available. 21. Device according to one of claims 17 to 20, characterized records that as a carrier for the ana lyter-recognizing and selectively binding complement molecules in addition to the typi rule microtiter material polystyrene preferably chromatographic sta stationary phases, such as adsorbent-filled columns, RP-18-like materials lien, blotting paper strips, membrane filter strips, the capillary flow proper have been used. 22. Device according to one of claims 17 to 21, characterized records that a planar three-electrode electrochemical electrode unimpeded contact with the different surfaces of the zones can find or the optical measurement alternatively between a carrier underground and the measuring zones can oscillate. 23. The device according to one of claims 17 to 22, characterized shows that, as a collecting and enrichment zone, there are very limited zones in the sample flow direction behind the first, analyte-specific binding Zone are created that consist of the relevant analyzer-recognizing and -bin end complement molecules that have free binding sites and bind the redox- or fluorescence-labeled analyte molecules and the right petitive measurement. 24. Device according to one of claims 17 to 22, characterized records that a lipophilic zone as collecting and enrichment zone used with oil or wax soaked surface or RP-18 material is, with ionic analytes of this phase still a lipophilic counter ion is added for ion pair formation. 25. The device according to any one of claims 17 to 22, characterized records that the catchment and enrichment zone for those in the first  labeled analyte-binding zone displaced labeled analyte molecules by a ge corresponding to the molar mass of the labeled analyte molecule chosen dialysis membrane is given, which is at a certain distance from the first zone is attached, and both stationary by means of evaporated Electrodes as well as dismantled can be measured electrochemically. 26. The apparatus according to claim 25, characterized in that the se dialysis membrane in the first place in the direction of flow lying surface immobilizes the analyte-binding complementary molecules contains. 27. The device according to one of claims 17 to 26, characterized records that the first analyzer-recognizing and binding zone and that there from separate collecting and enrichment zone within a test streak fen-like carrier with capillary forces. 28. Device according to one of claims 17 to 27, characterized records that this is a planar three-electrode measuring cell, which together is pressed onto these areas with a suitable base electrolyte, contains. 29. The device according to claim 28, characterized in that the Three-electrode measuring cell an ultra-microelectrode array made of a noble metal with single electrode diameters <10 µm. 30. The device according to claim 28 or 29, characterized in that that the working electrode or the ultra-micro-electro used for this the array is integrated into the different measuring zones. 31. The device according to claim 30, characterized in that the Reference and counter electrodes integrated in the surface of a wafer are.