WO1997021095A2

WO1997021095A2 - Process and device for carrying out highly accurate rapid chemical analyses

Info

Publication number: WO1997021095A2
Application number: PCT/DE1996/002351
Authority: WO
Inventors: Karl Cammann
Original assignee: Karl Cammann
Priority date: 1995-12-04
Filing date: 1996-12-04
Publication date: 1997-06-12
Also published as: WO1997021095A3; DE19545130C2; DE19545130A1

Abstract

The invention relates to a method and a device for determining the concentration of an analyte, analyte mixture or biochemical analyte-equivalent substance (titrant) contained in a sample using at least one selective reagent (titrator). Instead of the concentration-proportional intensity measurement known from prior art, the invention uses an absolute equivalent quantity measurement which can be miniaturised and can be read off with considerably greater accuracy. Measurement of the titrator quantity equivalent to the analyte concentration, i.e. of the chemical titrator equivalent to be measured, is reduced to a simple and rapid procedure for determining a location or line. The reaction in the reaction series, i.e. the point on a titration line, at which equivalent quantities of analyte and titrator are present is determined. Other process steps such as biochemical analyte-identification reactions or chromatographic separations can also be included.

Description

Method and device for high-precision chemical rapid analyzes

The invention relates to a method and a device for high-precision chemical rapid analyzes.

The state of the art in the field of so-called wet chemical analysis is, based on the absolute amounts of the substances reacting with one another (analyte with standard solution), almost the same as one hundred years ago when, for example, dimensional analysis was introduced as an absolute method of determination. In the so-called titrimetric methods (acid-base, redox, precipitation, complex, association, affinity, etc. titration) volumes of typically 10 to 100 ml are still processed and meanwhile also by means of automatic titrators titrated with integrated evaluation software. If the amount (chemical equivalent) of the added reagent (titrator) is given by the volume and the titer, the sample amount containing the analyte to be analyzed must be dosed differently depending on the analyte concentration in order to determine the volume titrator consumption in a Dosing accuracy to keep the range limited. Usually, the analyte concentrations in a titrimetric analysis should be within a decade. This usually requires appropriate dilution steps during sample preparation.

In the case of such wet chemical measurement analyzes, a correspondingly large amount of waste is produced in series analyzes, which waste must be declared as hazardous waste depending on the environmental relevance of the sample to be analyzed, the titrator or the equivalence point detection indicators or other auxiliary substances. In the over 100 million chemical analyzes carried out worldwide every day, quite a few are carried out as described above and therefore pollute the environment. A particularly drastic example is the determination of the so-called chemical oxygen demand (COD value), a more operationally defined parameter, as prescribed by the Water Tax Act. Here, according to a DIN method, it is determined how many substances in a sample are oxidized by a hot, acidic dichromate standard solution in a certain time unit. For this purpose, the excess of the oxidizing agent must be retained even after the determination, ie highly toxic chromate must be disposed of. If the chloride concentration is too high, a mercury salt must also be used as the chloride trap to bind this anion, which is also oxidized by the chromate but does not represent an organic load. ger can be added. These methods run with sample amounts of typically 20 ml and total volumes between 30 and 100 ml at concentrations corresponding to 0.1 normal standard solutions, which leads to milligram amounts of waste per analysis.

A miniaturization of the classic dimensional analysis can be used to reduce the waste. However, there are practical limits to a general miniaturization of classic dimensional analysis. Dealing with graduated volume measuring vessels with volumes of less than one milliliter is difficult and the current pipettes in the microliter range have errors that are too large and are designed accordingly in a complex manner. In a subsequent optical determination of the equivalence point, correspondingly small beam cross sections must be used, which leads to equipment problems with regard to the scattered light component.

Because the classic measurement analysis when using suitable titrators (original titer substances with the required "trace ability" for the mole) is an absolute determination method that does not require calibration using artificial or natural standards (certified standard reference materials), it generally also becomes a Control of any calibration or calibration solutions used for instrumental analysis. In contrast, most of the analytes of inorganic and organic types are today determined by so-called relative methods, which, however, have to be calibrated exactly. In almost all analytical-chemical relative methods, the intensity of a specific analysis signal (electromagnetic radiation, electrical current, resistance or potential, or another, analyte-proportional sensor or detector signal) measured and compared with that of a comparison sample with a known content. So far, only the last step of a chemical analysis, namely the measurement of the intensity of an analysis signal, has been significantly improved by the developments in equipment. In the medical field, in particular, there are machines which carry out a complete chemical wet analysis fully automatically (for example the Technikon auto analyzer method, which is characterized by a sample stream segmented with a gaseous intermediate space), which require these machines however a high expenditure on equipment.

Further developments are aimed at simplifying sample handling and automating routine analysis. An example of this is the flow injection analysis (EH Hansen, International Laboratory Vol. 15, No. 8, p. 14, 1986), which works analogously to the Technikon auto analyzer system, but does not use a segmented sample flow. Here, an automatic sampling valve (test loop) doses a certain volume of sample into a stream of a suitable carrier or transport liquid (carrier solution), which then mixes the sample with or without reaction or mixing chambers with all the necessary reagents lets a detector flow past. The unknown concentration is then calculated from the height or area of an analyte signal peak generated there. The advantage over the segmented methods is that any desired concentration gradients (dilutions) can be generated by the desired diffusion and the mixing of the sample segment with the transport liquid can. Another development in the field of wet chemical analysis are microprocessor-controlled table robot systems, which, however, only do the laboratory work automatically.

All known automated systems for wet chemical analysis have the disadvantages of the large expenditure on equipment and the relatively high consumption of reagents. For example, the automatic machines for clinical analysis are complicated and correspondingly expensive devices which require a correspondingly large parking space and also require a considerable amount of electrical connection power. As an alternative to the vending machines, the area of test kits has become established for quick analysis methods. For example, portable reagent kits have been developed which, by mixing with a sample aliquot in the milliliter range and mostly photometric analysis, should enable rapid analyzes. However, these systems also have the disadvantage that in many cases the measured samples represent hazardous waste, which is still produced in relatively large quantities. Micro-total analysis systems (μ-TAS) are possible for the latter area, but these are currently still in the development stage. Even if this extreme miniaturization of classic analytics were commercially available, the effort would be considerable due to the required micropumps and signal measuring devices and the device costs would be correspondingly high.

In response to these serious disadvantages of classic analytics, rapid tests based on dry chemical alternative methods have been developed in recent years (O. Sunday: dry chemistry: Analysis with carrier-bound reagents, Thieme Verlag, Stuttgart 1988). In these dry chemical methods, sample preparation, matrix separation and selective staining are carried out with a special reagent that recognizes the analyte on a planar support made of paper or plastic. In these processes, different layers with immobilized reagents or reagent mixtures are used. Test strips in which one or more test fields are arranged on a plastic strip were able to assert themselves in rapid medical analysis. These test strips are predominantly evaluated visually by comparing the reaction color of the test fields with a color scale which is often arranged on the label of the storage containers for these test strips. Elaborate and lightfast color printing processes must be used for the reference color scale.

The test strips only require the user to simply immerse them in the sample and then read off the concentration based on a color or an intensity of color. Such test strips are now available for a large number of analytes in the field of medicine, environmental protection, production control, etc. The problem with the rational test strips is that the time period for immersion and the way in which they are removed by stripping off excess sample volume is precisely prescribed and must be observed. The respectively analyzed sample volume is determined by a liquid absorbing material and is therefore very prone to errors. For this reason, only semi-quantitative results can be achieved. The subsequent visual analysis is not standardized. A number of Known can influence the quality of the results (W. Majunke, pocket paper volume 5, page 161, Springer Verlag, Berlin 1985). Different lighting conditions, disturb the inherent colors of the samples, differently accurate observance of the reading times and individually different color discrimination capabilities result in further error possibilities which stand in the way of a reliable rapid test.

An evaluation of the test strips using a reflection photometer would be more objective, but this does not lead to significantly more accurate results due to the individually possible influencing of sampling and sample handling. If the test strips are immersed for too long, they lose their special analyte-selective reagents in the sample and give incorrect results. If the rounding of the edge of the beaker is too strong for stripping, more sample remains on the test strip than was calibrated in. It is also difficult to standardize simply wiping with other aids.

The most common example of this analysis technique is the measurement of the blood sugar content.

To do this, a drop of capillary blood is applied to the surface of the test strip. The whole blood is filtered on the multi-layer test strip, separated from the red blood components and then spatially separated the analyte glucose with immobilized glucose oxidase is converted to gluconic acid and hydrogen peroxide. High molecular and cellular components remain and are wiped off the test field after the exposure time (one to two minutes). The amount of hydrogen peroxide formed is proportional to the glucose concentration. Hydrogen peroxide in turn functions as an oxidizing agent for a reaction between two organic molecules, which then form a proportional amount of a green-blue colored azo dye. In contrast to the above methods, the sample is transported here by means of the capillary action of the carrier materials. In principle, the measurement of the blood sugar content has thereby become as simple as a pH value measurement using an indicator paper.

All test strips, rods or tubes, which record a large number of dry chemical-based analytes that are important for the environment or the clinic area without recalibration, have, as already explained in part, a number of decisive disadvantages. In principle, the substance is recognized by a suitable, easily perceptible marking of the analyte by means of suitable aids (reagents). In most cases, using a quantitative, instrumental-analytical method, analyte concentration-dependent color intensity signals are measured using a wide variety of physico-chemical interactions. The different intensities of the basic colors also play a decisive role in the formation of mixed colors. A disadvantage of all substance concentration determinations which are based on the measurement of a specific signal intensity are the various disturbances which are associated with this. Regardless of the type of actual measurement signal (intensity of electromagnetic radiation, electrical potentials, electrical currents, conductivity, magnetic or electrical fields, etc.), changes in the excitation and output signal strength or sample matrix effects Contingent interference factors in the analyte-specific interaction with these excitation energies for incorrect measurements. As a rule, the sample must first be brought into an appropriate measuring device in which these measurements of the analyte-specific energetic interactions are carried out.

For example, a photometer can show instabilities in the excitation light source. The intensity of the reflected light depends on the intensity of the incident light. Therefore, in quantitative evaluations, the measurement must be based on the intensity of the incident light. The discoloration or color intensity to be evaluated can, for example, be negatively influenced by the matrix of the sample, as a result of which the slope of a calibration line varies and the method of standard addition must be used. The reflection photometers for evaluating certain test strips usually work at a fixed wavelength in order to make them compact and therefore portable. However, their area of use is thus limited to this spectral range. They cannot take advantage of the variety of known reagents and are not suitable for different indicators.

For reasons of increased selectivity, in addition to the restricted selective reagent, the dry chemical test strips can generally also contain other additives or auxiliaries (e.g. buffers, masking reagents,

Stabilizers, etc.) included. These substances eliminate cross interference from other substances in the sample matrix and stabilize the staining. All dry chemical methods have in common that the concentration determination (quantitative analysis) over 40 a color (ie wavelength-dependent) or color intensity evaluation is carried out. If one wants to measure different colors (eg in multi-analyte measurements), such a device must contain a monochromator and is complicated accordingly. Because of this expenditure on equipment, there are hardly any measuring devices which can objectively measure different colors of the different test strips. This considerably complicates the precise quantitative on-site multi-analyte analysis using test strips or sticks.

Since the dry chemical processes are relative processes, they are very susceptible to matrix. Everything that influences the colors to be evaluated, such as, for example, colored samples, suspensions, additional absorbent layers (for example precipitation, protein in the case of biological samples, etc.), which cannot be taken into account in the batch calibration of the manufacturer , leads to incorrect measurements.

In all rapid tests based on dry chemistry, the sample is transported only over minimal distances, which are overcome by diffusion and / or capillary forces. There is no possibility here of freely combining analytical-chemical operations (sampling, sample preparation, extraction, precipitation, outgassing, hydration, oxidation, reduction, neutralization, buffering, etc.) in any desired and application-oriented manner.

Based on the disadvantages of the prior art, the invention is based on the object of a method and a device for calibration-free marriage. AA mix or biochemical rapid analysis to create, which allow highly accurate dimensional analysis with low procedural requirements. In particular, the high reagent consumption of the analysis methods of the prior art is to be reduced and a simple integration of additional method steps supporting the analysis method is to be made possible.

This object is achieved in procedural terms by claims 1 and 26 and with respect to the device by claims 9 and 29. The respective subclaims relate to preferred and advantageous embodiments of the invention.

In the method according to the invention for determining the concentration of an analyte, analyte mixture or a biochemical analyte-equivalent substance (titrant) contained in a gaseous or liquid sample with the aid of a selective reagent (titrator), the known sample volume and the known chemical equivalent are used of the titrator, a series of spatially separated and quantitative titrator-titrand reactions are carried out, the individual titrator-titrand reactions differing at least in one of the two sizes of sample volume and chemical titrator equivalent.

This method is based on the implementation of a measurement analytical titration series, the equivalence point being set differently from reaction to reaction. The concentration-proportional intensity measurement known from the prior art is achieved according to the invention by a miniaturized replaceable and much more accurately readable absolute equivalence quantity determination replaced. The determination of the titrator amount equivalent to the analyte concentration, ie the corresponding chemical titrator equivalent, can be reduced to a simple and quick determination of the location or distance. In principle, the reaction in the series of reactions in which there are equivalent amounts of analyte and titrator must be determined.

The individual titrator-Titrand reactions of the reaction series can differ, for example, at least in that, with a constant sample volume, the titrator equivalent is changed incrementally (ie in known, not necessarily constant increments) from reaction to reaction or alternatively with a constant titrator equivalent the sample volume is changed incrementally. To increase the dynamic range of use or the detectable concentration range, both can also be changed simultaneously from reaction to reaction, that is to say both the sample volume and the amount of titrator used for the reaction.

The number of reactions required for the rapid test according to the invention depends on the application in question, the detection range and the desired accuracy. In principle, a single sample volume with an equivalent amount of the titrator corresponding to the relevant limit value is sufficient to determine whether the limit value has been exceeded. In order to increase the test accuracy and to reduce systematic errors, a statistically safe exceeding of the limit value can only be assumed if a large number of reactions 43 give a specific signal. This signal can be, for example, an optical indicator envelope or an electrochemical signal. The redundancy with regard to the number of reactions taking place increases the reliability of the rapid test extraordinarily without the analytical effort or, with corresponding miniaturization, the chemical waste increasing accordingly.

The present invention solves the basic problem of all so-called relative analysis methods and classic rapid tests which are based on a signal intensity measurement which is susceptible to interference, because according to the invention only an easily detectable absolute signal change (for example an indicator change) or beginning and to distinguish the end of a marked zone or an analytically relevant marking from a background. This makes calibration-free, absolutely measuring rapid tests possible for the first time, the accuracy of which only depends on the choice of gradual titrant dosage and / or sample aliquoting. Since the sharpness of the detection of a signal change in the dimensional analysis is known to easily amount to 0.1 relative% of the equivalent volume, extremely precise measuring systems can be constructed if, for example, 1000 micro-reactions are carried out in a suitable spatial arrangement . In this way, a whole decade of concentration of the analyte can be recorded with the same accuracy. In applications in which the expected concentration range of the analyte only varies within certain limits, for example in clinical analysis, the number of reactions can also be well below 100, depending on the desired reading accuracy. This means a localization speed and thus also an analytical-chemical accuracy in the defined range of 1%.

The titrator equivalent and / or sample volume are preferably incrementally changed between spatially adjacent titrator-titrand reactions. With such an arrangement of the individual titrator-titanium reactions, a signal change (for example an indicator change) takes place exactly between two reactions, the equivalence concentration of which is precisely known. The analyte content of the sample must therefore lie exactly between these two known values. It is also advantageous to arrange the reactions in a series of increasing or decreasing sample volumes or titrator equivalents, because if the distance between two neighboring reactions remains the same, the evaluation becomes clearer and is printed on the reading of a distance or an adjacent one Scale runs out. Since this is an absolute method due to the use of stable analytical reagents, with constant and known sample aliquoting, each reaction site can have an analyte concentration value in arbitrary units (mol / l, g / 1,%, ppm, ml / dl etc.) can be assigned. The amount of chemical equivalence of the analyte currently present in a sample can be read, for example in absolute mol units, simply and precisely, in accordance with the selected graduation of the titrator. This in turn has the effect that all reactions in which the analyte is present in a stoichiometric deficit show a different signal than those in which the titrator is present in a stoichiometric deficit. This redundancy compensates for random errors (for example a fluctuation caused by the inclusion of air bubbles). 4T The great and decisive advantage of the new class of analysis techniques according to the invention is that measurement signal changes due to non-analytical intensity influences (eg matrix-related signal influences), which usually falsify the signal intensity and thus the concentration information, are insignificant here and do not affect the measurement result. Because the absolute signal intensities (for example color intensities or colorations of the indicators) do not play a role in determining the concentration, it is immaterial if a reaction in the area before or after the equivalence point is disturbed. These reactions represent redundant systems and considerably increase the measurement reliability compared to traditional dry chemical test strips. Compared to a classic measurement analytical titration, there are decisive advantages in that you cannot over-titrate and automatically have an end point documentation. Reading and transmission errors are also reduced. Since, according to the invention, the signal intensity only determines the sensitivity with which the start and end of a measurement path or a location can be localized, intensity fluctuations, for example, of a light source used for reading, in contrast to the known opti ¬ evaluation methods, no falsification of the measurement result.

Sampling and volume dimensions are preferably carried out simultaneously for all titrator-titrand reactions of the reaction series. In this way, a uniform sequence of the individual reactions can be guaranteed. The method according to the invention can be carried out with any sample volumes and titrator equivalents. In addition to the use of sample volumes in the milliliter range and milliequivalents, the method according to the invention also makes it possible to advance into the micro- and nanoliter range and to use micro- or nano-equivalents of the titrator. This stems from the fact that, as will be explained in more detail later, the method according to the invention can in particular also be carried out using microstructured test kits. Appropriate micro systems can be used to penetrate the areas of ultra-micro analysis. Chemical conversions on a microliter and nanogram scale save chemicals by a factor of 1 million compared to common laboratory practice. The waste load is correspondingly low. Because of the elimination of thorough vessel cleaning steps, less detergent is used per analysis. Because of the reduced reagent consumption, the method according to the invention can be carried out more cheaply than methods of the prior art.

The result of the concentration determination using a plurality of titrator-titrand reactions can be determined in various ways. In the simplest case of the additional use of optical indicator reagents, the concentration can be determined visually. Extreme miniaturization, however, reaches the limit of readability with the naked eye. According to the invention, an evaluation device is used here which shows the reaction result greatly enlarged. An advantageous arrangement arises from the fact that a strongly magnifying projection, similar to a hand slide viewer, is carried out. λl leads. If only the position of the signal envelope is important, simple devices operating with daylight can also be used. According to the invention, such a determination of the concentration can be advantageously measured automatically and in absolute concentration units by means of a suitable so-called barcode reader, which has to detect differently colored areas, by means of detectable electromagnetic radiation. With such an arrangement, the bar code reader only counts the number of reactions up to the equivalence point reaction, which amounts to an absolute, digital analysis. According to the invention, the reading of the analytical equivalence concentration (location determination) is not only limited to the visible range of the electromagnetic spectrum. Thus, when using indicators which fluoresce in the range from 800 to 1100 nm, implantable medical test devices which can be read from the outside can be implemented. According to the invention, further equivalence (end point) determination methods can also be used. For example, such a determination can also be carried out electrochemically (for example, amperometric or potentiometric) by means of an individually addressable electrode array which is brought into contact with the individual reactions.

In addition to the indicator reagents already mentioned, other substances such as auxiliaries such as buffers, masking or complexing substances, stabilizers or reaction accelerators can be mixed with the titrator or the sample before the titrator-titrant reactions or only during the titrator -Titrand reaction are added. After the sampling and before the titrator-titrand reaction with the sample volumes to be analyzed, additional neutralization, complexation, oxidation, reduction, separation or gas washing steps and / or sensitivity and selectivity-increasing chemical and / or biochemical analyte recognition reactions and / or chromatographic or electrophoretic separation operations are carried out. Separation operations can be carried out, for example, with the aid of filters or gas-permeable membranes, amplifications, for example, using the PCR method. Separations which can be used are, for example, gas-liquid or liquid-solid separations, separations of a chromatographic type (for example GC, LC, IC, RP-LC, gel, paper, thin-layer, affinity chromatography) or extractions.

A device according to the invention comprises a sampling module with a plurality of sampling devices and a titration module with a plurality of spatially separated reaction areas, the individual sampling devices being loaded with different sample volumes and / or different in the area of the individual reaction areas immobilized chemical titrator equivalents and the respective sample volumes and respective titrator equivalents are known.

Advantageously, incrementally increasing titrator equivalents are immobilized in or on the preferably linearly arranged reaction areas. Such an arrangement allows the result of the concentration determination to be read quickly. When using indicator reagents, λ, shows a clearly visible envelope between two reaction areas, the equivalence concentration of which is precisely known. The analyte content of the sample must consequently lie between these two equivalence concentrations. The determination of the concentration can be further simplified by applying a scaling in the area of the reaction areas. The titrator can be immobilized, for example, as a dry chemical substance or as a gel, suspension or as a drying liquid in the area of the reaction areas. If the sampling module and titration module are integrated, the titrator can also be immobilized in the sampling devices themselves, if necessary, neglecting the intrinsic volume.

To simplify the process sequences according to the invention, the spatial arrangement of the sampling devices in the sampling module can be correlated with the position of the reaction areas. For example, both sampling devices and reaction areas can be arranged in the form of congruent matrices.

The reaction areas can be designed in different ways. It is thus possible to design the reaction areas as depressions in the titration module, each with the same or different capacities. For example, given constant sample volumes and the same capacity of the wells, different titrator equivalents can then be arranged in these reaction areas. Alternatively, it is conceivable in the case of depressions with, for example, progressively increasing capacities, these with different volumes 2C to fill a titrator-indicator mixture, for example. In both alternatives, the chemical titrator equivalent immobilized in the area of the respective reaction area increases from deepening to deepening. The shape of these depressions (reactors) is irrelevant as long as the diffusion of the sample is not influenced too much.

A decisive advantage of the device according to the invention for determining the concentration is the simple miniaturization. Using modern structuring techniques, recesses with a capacity in the micro and nanoliter range can be created in suitable materials such as silicon or plastic. Microstructured devices according to the invention allow drastic savings in terms of the chemicals used and thus lead to a significantly lower waste load. The disposal costs decrease accordingly.

Another possibility for defining miniaturizable reaction areas is provided by thick film techniques such as screen printing. The reaction areas structured in this way can have the form of rings or honeycombs. However, not only the reaction areas can be defined by means of the screen printing technique, but also the immobilization of the titrator can be carried out. Quantities of the component to be immobilized increasing from reaction area to reaction area can be applied, for example, by a multiple printing process. For this purpose, the component to be immobilized is preferably in the form of a gel capable of drying. For example, paper or plastic foils are suitable as substrates for such film techniques. After completion of the A further, preferably transparent, layer can be applied to printing processes to define the reaction areas and to immobilize the titrator. For example, the appropriately printed substrate can be enclosed in a heat-polymerizable film.

Although the reaction areas are preferably arranged in a linear manner, a two- or three-dimensional matrix arrangement or a circular, spiral or helical arrangement of the reaction areas on or in the titration module is also conceivable. In the case of a circular arrangement, for example, the concentration can be determined analogously to reading a clock. To achieve a large dynamic

Measuring range and a high degree of accuracy of the measurement analysis and an associated large number of required reaction areas, the reaction areas and the associated sampling devices can be designed as a two-dimensional, planar arrangement (for example 30 rows and 30 columns in credit card format). Here, the analytical reagent positioning and grading can proceed in such a way that, for example, horizontally there are larger concentration jumps between the individual reaction areas and the vertical jumps divide these larger jumps into smaller ones. The micro-system technology also allows three-dimensional structures, for example cubes with two-dimensional arrangement of the reaction areas in question, to be designed. In this way, six concentration decades of an analyte can in principle be covered by the six sides of a cube or six analytes can be determined in parallel. During production, the volume of the reac- tion areas per side of the cube, advantageously change the concentration of the analytical filling solution per side in accordance with the desired dynamic measuring range.

The individual sampling devices of the sampling module are preferably designed as cavities such as pipettes or capillaries with the same, different or variable capacities and are filled with known sample volumes. The sampling devices can also be designed as sample collection devices with, for example, capillary feed lines for filling.

The sampling devices can be formed as recesses arranged in matrix form with dimensions in the micro or millimeter range in a material with hydrophobic surfaces. The hydrophobic surfaces ensure precise sampling, as excess sample quantities after filling the

Sampling devices do not stick in the area of the openings. The hydrophobic surfaces also ensure the complete quantitative transport of the sample volumes to the reaction areas. The quantitative transport is therefore important because the sample volume reacting in the reaction areas must be known exactly for the highly precise determination of the concentration.

For filling the sampling devices, immersion in the liquid or gaseous sample, for example, is sufficient so that a defined and known sample aliquot is subsequently produced by capillary forces, diffusion, gravity, centrifugal force, mechanical piston force, pressure differences or a transport liquid can reach the reaction areas. The penetration of the sample into the sampling devices must be controllable for the precise measurement of the sample volumes. For this purpose, the sampling devices should be closable on the side facing the sample. This can be done using a wide variety of valves or micro-valves (actuators), which can be moved, for example, electrically or magnetically. An advantageous, energetically passive form is that of a non-return valve at the entrance of the sampling device, which is closed in that a swelling substance previously attached to the entrance firmly closes the non-return valve. The use of self-dissolving membranes is also conceivable. Passive magnetic valves, which can be controlled from the outside by means of external magnetic fields, are an advantageous solution for gaseous samples, which usually also require larger pipette spaces. The contact time, ie the

The time of diffusion of the analyte through reagent layers of the test arrangement can be set precisely. A further preferred form of the valve-like control uses displaceable spacers with suitable cutouts between at least two interfaces with corresponding openings for filling and emptying the measurement volumes formed by the cutouts.

For the analysis of gaseous samples, the sampling devices can be designed as diffusion tubes and can be connected to a module containing a collection phase for gaseous analytes. The gaseous analytes are chemically bound in the module and subjected to further process steps after a certain collection time. IN THE

The concentration determination method according to the invention can be simplified by integrating the sampling and titration module (or the corresponding method steps). The sampling module and the titration module accordingly form a unit, the reaction areas being formed within the sampling devices. Here it is particularly advantageous if the sampling devices can be closed on the side facing the sample after the sampling.

A particular advantage of the device according to the invention is that by combining modules which are designed to match one another, arbitrarily complex rapid test arrangements can be put together. The individual modules are preferably designed in such a way that channels can be opened and closed among one another by means of displacements which are planned according to the invention. The individual modules can be in direct contact (planar surface). However, thin, displaceable spacers (e.g. made of Teflon) with or without volume-defining recesses can also be placed between them as a seal.

A preparation module is preferably arranged between the sampling module and the titration module. The sample volumes are transported from the sampling devices in the sampling module through the preparation module to the reaction areas of the titration module, preferably on a micro or millimeter scale. In the preparation module, steps such as oxidation, reduction, gas expulsion, electrophoretic or chromatographic separation, chemical or biochemical analyte detection, amplification or separation can be carried out. The preparatory 2 poule can be configured, for example, as a permeable membrane, as a filter, as a chromatography column or as a spacer between two planar interfaces, which has flow channels or diversions.

As a selectivity-increasing analyte recognition reaction in the preparation module, biochemical reactions which operate according to the key-lock principle (for example enzymatically or immunologically with redox derivatives, ELISA method-analogous or amplifying, for example by substrate) Cyclization or by means of PCR technology) preceded by the analytical quantification. A stoichiometrically released, analyte-equivalent, i.e. quantitatively strictly proportional further substance determined by the method according to the invention, which requires a 100% metabolism of the analyte in the first two types of biochemical reactions.

Antibody molecules that are not saturated at the binding site can also be used as titrators for the analytes in reaction areas in the form of affinity columns of different but known analyte binding capacity. Here the breakthrough concentration corresponds to the equivalence point. The selective mixtures of known gas test tubes, for example showing organic compounds, can be used as indicators. Alternatively, affinity columns can also be constructed in which the analyte-specific antibody molecules introduced there are 100% saturated at the corresponding binding sites with a derivatized form of the analyte, but the actual analyte has a much higher affinity constant than its deri vat. According to the invention, the derivatized molecular region of the analyte should be detectable by means of measurement analysis methods. For example, a stable redox group (eg ferrocene, ascorbate, quinone) in the uniform redox state can be covalently attached to the analyte molecule. This redox group is displaced stoichiometrically in proportion to the analyte content from the affinity column in the course of the parallel reactions and, as explained above, can be detected precisely in equivalents (mol units) in the reaction areas using corresponding redox reagents. Of course, the affinity column can also be combined with the sampling device (eg pipette). Examples of this are so-called capillary fill devices.

The individual modules of the overall device can be made from recyclable materials, such as plastics (plexiglass, polypropylene, PET, polycarbonate, PE or other types that can be structured precisely), silicon, ceramic (green tape technology), paper or paper-like Chen materials exist. It is possible to apply the modules as planar devices on a flexible substrate (plastic film, hot-melt film, paper), which is rolled up and brought into contact with the sample, for example, and rolled out to read off the analyte concentration in question.

The individual modules are, for example

Thick film techniques such as screen printing, structured or microstructured using injection molding, CNC technology or LIGA processes or using semiconductor structuring processes such as photolithography. In particular through the modern technology of photolithographic see methods of micro-system technology, both the most precise micro-pipettes and the micro-reactors with precisely controlled volumes can be produced easily, inexpensively and with very small deviations. This also applies to the LIGA technology, which is not limited to silicon, but also enables inexpensive injection molding solutions in plastic. With dosing accuracies in the% range, thick film technology can also be used. The titration module is preferably designed as a planar support with a layered (laminate) structure (eg paper strips in credit card format). However, a shell-shaped or tubular, circular, spiral, spherical or cube-shaped structure is also conceivable. An advantage of semiconductor or LIGA technology is the fact that the individual modules can be produced together in a single work step. The micro-system technology also enables precise metering and positioning of the analytical reagent. The analytical reagent does not necessarily have to be a stable reagent.

The individual function modules are preferably structured in such a way that the titrant can penetrate the preparation module or the titration module by mutually displacing the modules or an intermediate spacer with corresponding recesses. Moving can open and close channels. However, pistons can also be provided which displace the sample volume from the sampling devices in the direction of the reaction areas. The pistons can be integrated in the sampling module or in a modular module that is separate from the sampling module Be trained. As an alternative to this, a multi-pipette block (sampling module) with different pipette volumes and injection openings can be produced, in which all pipettes are filled by injection of the titrator (including the indicator and, if applicable, of auxiliary substances) and then, after being placed on appropriately designed micro-reactors, with constant and known amount of sample by means of a displacement of spacer valves the outflow of the different titrator amounts takes place in the reactors.

For the above-mentioned embodiments of the device according to the invention, spacers, sliding layers or separating foils can advantageously be located on or between the individual modules.

In a further embodiment of the method according to the invention for determining the concentration of an analyte, analyte mixture or a biochemical analyte-equivalent substance contained in a sample with the aid of a selective reagent, a known sample volume is reacted with at least one titrator immobilized with a known chemical equivalent density and the titrant concentration is determined by measuring the geometric dimensions of the partial region of the immobilized titrator in which a specific titrator-titrand reaction taking place quantitatively and quantitatively has taken place. The equivalent density here denotes the immobilization density of the titrator, for example in mol / cm or mol / cm ² . In this method too, the analyte concentration is not determined via signal intensities susceptible to interference, but preferably by dimensioning one one-dimensional (linear) geometric range or reading a scale.

After a known sample volume has been applied to the immobilized titrator, the sample solution moves in the layer of the immobilized reagent due to, for example, capillary, heavy, piston stroke or centrifugal forces and is successively titrated (more precisely: back-titrated, since first the excess of the titrator must be eliminated by the diffusing amount of analyte), the stretch of the excess analyte then showing a color when an optical indicator is used. With a known immobilization density of the titer, the length of the stained section can be used to infer the analyte concentration. As already mentioned above, the color itself or the constancy of intensity of the specifically stained zone is irrelevant for determining the concentration, since the concentration is determined by measuring the distance or area. The color intensity only determines the sensitivity with which the beginning and end of the measuring section can be recognized. Only when the analyte has completely reacted on its way with the titrator fixed there, is it selected with a selective one

Coloring cocktail can no longer be colored. Consequently, thin-layer chromatography can also be converted according to the invention into a simple-to-measure absolute method if diffusion barriers prevent the diffusion of the analyte or analytes transversely to the direction of flow of the mobile phase and thus only allow diffusion in one direction. Suitable diffusion barriers are all materials with a diffusion coefficient which is negligible with respect to liquids, for example solids. The novel, Miniaturizable multi-analysis methods significantly expand the traditional thin-layer chromatography method with regard to the accuracy and simplicity of the evaluation.

Most of the explanations regarding the first method for determining the concentration can also be transferred to this second method with a continuous reaction space. In the present case, for example, the length or area measurement of the titrator-Titrand reaction can also be carried out visually, electrochemically or by means of optical evaluation methods such as barcode readers or analogously to CD players. The width of the staining zones can be determined using a barcode reader. Fluorescent zones (active fluorescence) or zones with extinguished fluorescence (negative fluorescence) can also be measured.

According to the invention, the sample volume can be subjected to oxidation, reduction, gas expulsion, electrophoretic or chromatographic separation, chemical or biochemical analyte detection, amplification or separation and then brought into contact with the immobilized titrator. In this regard too, reference is again made to the previous statements.

A device according to the invention for carrying out the method comprises a carrier on or in which at least one titrator channel is arranged. At least one titrator substance is immobilized in the area of this titrator channel. The titrator channel preferably has a linear shape. Circular or spiral channels are also conceivable. M

The titrator channel length is typically 1 to 20 cm. The dimensions of the channel in the running direction can be varied, which increases the detection area. The titrator channel is preferably a capillary channel. The capillary action of narrow channels increases the flow rate of the eluent and thereby considerably shortens the analysis time (compared to conventional chromatographic methods). Typical titrator channel diameters are in the μm range.

The titrator channel can be preceded by a preparation channel in which the process steps already mentioned, such as separations, oxidations, etc., take place before the actual titration. The sample is then only brought into contact with the titration channel, passes through it and finally reaches the titrator channel, in which the actual titration then takes place. With longer separation distances, the amount of sample taken may not be sufficient to reach the determination zone after the separation zone. In this case, the test device is dipped into a solution after stripping off a wetting layer, which solution then represents the chromatographic mobile phase. The latter is preferably transported to the determination zone by pure capillary forces or by piston stroke. There, the analyte separated from the other constituents is titrated two-dimensionally, as previously described (quantification zone for the absolute determination). Since the analyte flows horizontally through the channel filled with the titrator, similar analyte concentrations can also be used with a constant cross-sectional volume. According to the invention in non-electrophoretic separations Adjust the grain size of the stationary phase to the channel cross-section. Channels in the μm range require a nanoporous stationary phase for optimal function. The channels can be operated open or covered. In the case of a dense cover, the stationary phase expediently fills the entire cavity formed by the channel and the cover. In a further embodiment, the mobile phase dissolves the substances on the top of the cover for substance detection and distributes them by diffusion over the entire cross section.

The sample volume can be loaded into the titrator channel via a sample channel, which is more in contact with the at least one titrator channel. The sample can be transported via capillary forces, hydrostatic or other pressure (e.g. by a piston movement). A cover can be provided for the titrator channel, in which a sample channel is formed. The sample channel can be a capillary channel.

An advantageous embodiment of the invention provides that a plurality of the same or at least partially different titrators in each

Area of separate titrator channels are immobilized and the individual titrator channels are arranged side by side or one after the other. With such an arrangement, several analytes can be detected simultaneously in one sample or a large number of samples can be examined simultaneously for a specific analyte. In the case of a large number of titrator channels arranged side by side or one after the other with different or identical titrator substances, the position of the optically marked strip provides information About the type of substance (different titrator substances) or about the respective concrete sample (same titrator substances) and the stained or non-stained section is proportional to the concentration (quantitative analysis). The geometric shape of the chromatographic channels is less significant. Semicircular, oval, V-shaped, trapezoidal and rectangular depressions (channels) of constant depth on a wide variety of substrates (e.g. glass, ceramic, silicon,

Plastic, metal, etc.), which can be filled with the common and known chromatographic stationary phases. The smaller the channel cross section, the more channels can be accommodated on a carrier with given dimensions.

Multi-channel devices can be read out, for example, by means of a barcode reader. After a start signal at a constant line scanning speed (scan rate), a barcode reader receives a further signal, the time delay of which represents the type of substance and the time duration (stripe width) depends on the concentration of the analyte. Further staining zones correspond to further samples or other substances which can be measured simultaneously with or without additional selectivity-generating reagents. According to the invention, this evaluation can also be carried out in special measuring devices which move the separating sections past an analog optical read head analogously to a CD player. In this way, beams with many channels in particular can be measured very quickly. In the case of circularly arranged channels, device structures very similar to a CD player, which evaluate thousands of analyzes in a few seconds, are possible. Circular channels However, especially in the case of electrophoresis, special start and end zones with electrolyte contact for transmitting the electrical field are required.

The substances to be determined in parallel are expediently optimized according to the particular application. In clinical chemistry, for example, the so-called electrolytes are determined in parallel. Alternatively, for example, glucose with lactate, creatinine, urea and uric acid can be determined simultaneously if the oxidases in question are immobilized as reagents, which generate a quantity of hydrogen peroxide corresponding to the analyte stoichiometrically using the co-substrate oxygen. To determine the absolute concentration of the amount of hydrogen peroxide, this is "two-dimensionally titrated" with an immobilized reducing or oxidizing agent based on a redox titration.

The carrier for devices according to the invention can be structured or microstructured using thick film technology such as screen printing or using the LIGA process or using semiconductor structuring processes such as photolithography. If silicon is used as a support for the channels, a very large number of narrow, preferably parallel channels can be produced very simply and uniformly by photolithography and anisotropic etching using the known techniques of microelectronics. The well-known LIGA technology can be used for plastic carriers, which enables the same. Structuring by means of injection molding or CNC technology is also possible. Quartz, glass, metal and other solid bodies can also be scratched or etched accordingly using known techniques. But there are also circular shaped channel arrangements possible. In theory, well over ten parallel analyzes can be carried out per cm of beam width.

Some exemplary embodiments according to the invention are described in more detail below with reference to figures. Eε show:

FIG. 1: a planar, test strip-shaped device made of a transparent plastic for carrying out the method according to the invention,

FIG. 2: the structure of a titration module and a sampling module with variable but known volumes of the sampling devices,

FIG. 3: a device for the simultaneous analysis of a sample for different analytes,

FIG. 4: possible arrangements of the reaction areas on or in a titration module,

FIGS. 5, 7, 8, 9: the construction of further devices according to the invention with a large number of reaction areas,

FIG. 6: a device for the simultaneous analysis of a sample for different analytes or for the simultaneous analysis of several samples and

Figure 10: the sample transport in a device according to the invention by means of piston technology. The following exemplary embodiments primarily consider microstructured systems which, owing to the low consumption of chemicals, are particularly suitable for the process according to the invention.

In embodiment 1, the structure of a modular device according to the invention with electrochemical indication is outlined.

The structure consists of a planar, test strip-shaped arrangement made of a transparent plastic with dimensions of approximately 1 x 0.5 x 10 cm (W x H x L). Such an arrangement is outlined in FIG. 1. The arrangement consists of a sampling module 1 with a one-dimensional matrix of sampling devices 6 according to the invention in the form of micropipettes with a constant and precisely known volume in the microliter range. In this example, the scaling 5 of the dimensionally analytical micro or nano equivalents of the titrator, which is located in the reaction areas 4a, is attached to the sampling module 1 due to the transparent material. However, it can also be arranged on the underside of the titration module 2. The micro-capillaries 6 are sealed with a separating film 7 at the bottom. An arrangement with spikes 3 and connecting channels to the individual reaction areas designed as microreactors 4a with volumes in the microliter range allows the separating foils 7 to be pierced quickly. The volume of the microreactors must be large enough to be able to take up the sample volumes without loss. In the case of other types of devices without spikes, self-dissolving membranes made of non-interfering compounds such as dextrin, sugar or salt can be used instead of the penetrable membrane. FIG. 5 shows a further embodiment of a device according to the invention.

A possible mechanism for the sample transport by means of a piston is outlined in FIG. The exploded view shows views and corresponding cross sections of an individual titration reactor of a modularly constructed analysis device having a plurality of reaction areas. The reaction area 4 is designed as a recess in a titration module 2. The dissolved or immobilized titrator substance and possibly an indicator are arranged in this recess. A separating film 7 and a sampling module 1 are arranged above the titration module. The sampling module 1 has a large number of sampling devices 19 configured as sample collection devices, which are connected to capillary feed lines. A plate 18 is arranged above the sampling module 1 and has a number of thrust pistons corresponding to the number of sample-taking devices 19. The thrust pistons have thorns. Between the plate 18 and the sampling module 1 there is a spacer 17 which has recesses for guiding the piston and also serves as a cover for the sampling module 1. The sample volumes in the sampling devices 19 are conveyed into the reaction areas 4 in that the thrust pistons force the samples out of the sampling devices 19 downwards and at the same time pierce the separating film 7 with the spikes attached to the individual pistons. The individual reactions can be evaluated in different ways. Instead of a visual equivalence point determination with the aid of a suitable indicator in the microreactors, it is also possible, for example, to carry out an electrochemical indexing which is not shown in the figures. In the case of an electrochemical indication of the dimensional analytical titration in the isolated micro reaction rooms by means of corresponding micro electrodes, the microchemical, dimensional analysis conversions take place in a module located above. In this case, indicators can be omitted. Rather, the sample volumes flow after the analytical reaction in a separate module into the reusable measuring module with a number of electrochemical measuring cells corresponding to the number of micro-reactors. Each of these measuring cells can be electrically contacted via contacts embedded in the floor and measured in terms of conductivity, pH value or redox value. The electrochemical equivalence point reader is reusable after rinsing. It is completed by a precise fitting of the reaction module, the sampling module and a sample preparation module possibly arranged in between to form a rapid test arrangement.

FIG. 2 outlines the structure of a sampling module with variable but precisely known volumes of the capillary pipettes 6. The wedge shape is not to scale. In addition to the variable length of the capillary pipettes 6, their diameter can also be changed so that the aliquotization can be changed by a factor of 100. If the capillary pipettes 6 are too long, however, the production technology of the LIGA method is not possible. Here the interior tubes are drilled, which can be done, for example, using classic precision engineering, or using a laser. The sampling module with variable aliquotization shown in FIG. 2 is reusable. To do this, the module is flushed with the new sample using a syringe. For this purpose, the two cover plates arranged on guide rails and provided with connecting capillaries 8 connect the individual capillaries 6 alternately below and above in such a way that the capillaries 6 can be filled by means of a single flush. Due to the variable sample aliquotization, the Sanne11 test system allows the dimensionally analytical titration equivalents in the micro-reactors 4b to remain constant in the titration module. FIG. 4 shows further arrangements of reaction areas 4a, 4b for receiving different or the same chemical titrator equivalents.

The individual micro-reactors of the device according to FIG. 2 are filled with the individual sample volumes simultaneously by the two cover plates provided with connecting capillaries 8 being shifted laterally. The micro-reactors 4b can also be accommodated on a paper-like support.

A rapid analysis with variable sample volume and constant titration equivalent in the micro-reactors comprises the steps of immersing the sampling module in the sample, placing it on the electrochemical measuring module with the interposition of a suitable foil with the one to the rich constant immobilized amounts of the titrator, opening the sample channels and the sealing of the analytical reagent and the rinsing into the measuring cells, the measurement of the measuring cell matrix to determine the location of the greatest change in the measuring signal and the reading of the equivalence concentration of the analyte present there.

FIG. 7 shows a further variant of the device according to the invention. The micro-capillary pipettes 6 (shown in a top view and a side view) can be connected to the micro-reactors 4 a by precisely fitting displacement. FIG. 7 also shows a three-dimensional representation of a micro-reactor.

Figure 8 outlines the structure of a composite arrangement of two modules. The sampling module 1 consists of a ceramic injection molding with drilled pipette channels. Immobilized reagents 12 for sample preparation are housed in a corner of the channels. The surface 13 is hydrophobic. The carrier material is closed off by a base plate 14.

FIG. 9 shows schematically the top view and side view of a further module 15 with micro-chambers 16 still closed, which can be opened if necessary.

The embodiment 2 discussed next describes the determinations of the acid or base concentration by means of a system configuration from individual reactors.

On a planar carrier from an inexpensive, According to FIG. 1, the material that can be structured using LIGA technology is a linear matrix of micro-reaction chambers with increasing volume and associated capillary-filling pipettes with overall dimensions of 10 × 1 × 0.5 cm (L × W × H) ) realized. In order to simplify a reproducible and automatable filling of the microchambers (reactors with filling volume on a micro and nanoliter scale, diameter typically 0.1 mm), these can also be produced with open-bottom chambers, which then, after filling, using a wide variety of techniques be closed with an optically permeable base plate. Filling with amounts of the titrator corresponding exactly to the chamber volume (in the acid determination, a lye is used as the titrator, and an acid is used in the lye determination) is carried out, for example, by means of a dispenser. However, the filling can also be carried out by immersion filling in a standard solution, preferably in gel form, of a low-volatility alkali or acid, to which a suitable pH indicator is added. Examples of immobilized alkalis are, in addition to the known basic alkaline substances such as alkali carbonates, also substances, for example strongly or weakly basic ion exchangers, which can bind protons on their surface (for example by means of amino groups). Accordingly, examples of immobilized acids are, in addition to the classic acidic Urtiterub¬ substances such as hydrogen sulfate and phosphate, among other strong or weakly acidic ion exchanger (for example with

S0 ₃ H groups). Alternatively, titrations can of course also be carried out on a nanoliter scale if the titrator is only covalently attached to the differently sized chamber surfaces. If such a test device is immersed in the sample to be measured, then at the same time all, depending on the desired graduation, more or less numerous, micro-pipettes are filled by the capillary action. Active filling of the pipettes by a controlled piston stroke is also possible (cf. FIG. 10). After the contact of the test device with the sample to be measured has been prevented, the further transport of the sample into the micro-titration chambers located below in each case in exemplary embodiment 2 is started by either piercing a sealing film or the micro-reactors in the titration module a self-dissolving layer are covered. As an alternative to this, the titration can also be triggered by a controlled displacement of the sampling module or a spacer provided with cutouts by opening suitable channels. The analyte then reacts with the precisely known amount of the base or acid standard present in the micro-reactors, with the indicator color changing only there depending on the acid or base concentration in the sample. where the equivalence has been exceeded. The absolute concentration of the analyte (here acid or base) can then be read off precisely on a scale attached to the side. The sample can also be transported by a mechanically or otherwise initiated capillary action or by immersion in a development liquid with or without auxiliary reagents. It is also conceivable to fill the micro reaction chambers by means of vacuum or excess pressure. By choosing a suitable and uniform pH indicator which corresponds to the acid or base strength (pK value) to be determined, there are uniform staining areas (titers) With the micro-reaction with or without a stoichiometric analyte excess), which can also be evaluated automatically as a digital yes-no answer (with regard to reaching the end point). The method according to the invention and the device according to the invention thus ensure a high level of analysis accuracy.

In the analysis method of the second embodiment, the classic titration of an acid to be determined with an alkali (or vice versa) is converted into a series of parallel and separate micro titrations of controlled, varied titrator equivalents, which are changed by the type of The arrangement of the micro-titrations enables a digital or distance-based evaluation similar to that of the volume reading on a burette. The advantage according to the invention lies in the fact that only the position of the still stained zone with the highest immobilized alkali content has to be determined for the exact determination of the concentration, all other information is therefore redundant. It is also advantageous that the difficult and erroneous estimation of the color intensity of the colored zones no longer has any influence on the measurement accuracy. In the case of pH measurement, which becomes very imprecise, especially with strong acids and lyes, this "area titration method" is therefore much more precise. The analysis method according to the invention is evaluated in a manner comparable to LED light chains for displaying the intensity of electronic signals.

According to the invention, the titration modules that are precisely manufactured in terms of manufacture for an absolute determination of the concentration of acids and alkalis can also be used for gas analysis. For example, carbon dioxide can be reacted with known amounts of sodium hydroxide solution in a micro gas scrubber module. The decrease in the NaOH concentration can easily be determined below. Analogously, ammonia can be washed out and determined using HCI.

In embodiment 3, the absolute series determination of acids and alkalis is considered by means of a system configuration according to the invention (titration in reaction channels).

If a large number of samples have to be checked for their acid or base concentration, the preferred embodiment of the invention shown in FIG. 3 differs from the two previous exemplary embodiments. For each analysis there is a sample application device 9 on a microstructurable carrier, which is connected to an underlying microchannel 10 of defined dimensions. The entire cross section of this channel is completely filled with the standard titrator reagent, to which a suitable pH indicator is also added. The base plate can again be optically transparent. In this exemplary embodiment, the analysis sample is pressed by means of an exact microliter syringe over the feed device into the channel which functions as a continuous micro-reaction chamber. The sample plug is pressed into the channel by means of a short rinsing with the same or a second syringe or with an integrated piston. The neutralization reaction then continues in the channel evenly filled with the immobilized titrator until the stoichiometric excess of titrator is reduced. The HS

Concentration is determined, as in the two previous exemplary embodiments, by measuring a microchannel section with a known chemical equivalent density of the titrator. The absolute result could consequently be read off on a scale arranged next to the microchannel. In FIG. 3, markings 11 are shown, which, when the analysis result is continuously read out, for example by means of a barcode reader, allows the respective measurement result to be assigned to the analyte under investigation.

As an alternative to the procedure described, it is also possible first to fill all sample application areas with a sample volume on a microliter scale and then to immerse the entire multi-analysis device in a solution in order to bring about a sample transport through the reaction channels by capillary forces . As shown in FIG. 6, the sample can also be transported on impregnated blotting paper. To stabilize the surface titer, the embodiment described can also be sealed in a hot-melt film with the aid of a suitable spacer.

The following exemplary embodiment 4 outlines the determination of an analyte with redox properties by means of a "two-dimensional redox titration".

In the devices according to exemplary embodiments 2 or 3, the reducing agents required for this purpose are immobilized in the micro-reactors when an oxidizing agent is determined. Conversely, when determining a reducing agent, a suitable oxidizing agent with known chemical equivalents is used. density arranged there. Known redox indicators are used as indicators. In some cases, the redox titrator that has been chemically converted or is still present in excess also shows itself. Examples of this are permanganate, chromate, iodine, etc. Oxidizing agents with a standard potential more positive than + 0.35 volt (GKE) can be used with immobilized potassium iodide and approximately strength (eg 0.05 M KJ with 5% strength) very well with both Absolutely quantify devices if a reducing agent with a more reducing effect as titrator is immobilized together therewith.

Hydrogen peroxide is very often measured in biochemical processes. This is possible with great accuracy using the exemplary embodiment described here. Free hydrogen peroxide, which generates elemental iodine, is only present after the equivalence point of the reaction with the immobilized reducing agent has been exceeded. The elemental iodine produces an intense black color with the starch. All reducing agents that can be immobilized in the micro-reactors, such as titanium (III), iron (II) salts, arsenite, thiosulfate, oxalate, ascorbic acid, etc., can also be used as reducing agents together with suitable other redox indicators. An absolute quantitative determination of hydrogen peroxide is very interesting for all biosensors based on oxides.

In the quantitative determination of analytes with reducing properties, oxidizing agents with a correspondingly more positive redox potential are immobilized in the micro-reaction chambers. Particularly advantageous, simple design Guide forms are obtained if a self-indicating substance, such as potassium permanganate or Ce (IV) salts, which fluoresce in UV light, is used for this purpose. Chromate also changes its color from yellow to green.

Embodiment 5 deals with the determination of inorganic phosphate by precipitation as aluminum phosphate with a precipitation reactor according to the invention.

In this exemplary embodiment, aluminum sulfate is used as the titrator in an acidic environment. The free aluminum ions with immobilized Morin give an intense green fluorescence under UV radiation. If the analytical aluminum ions are increasingly removed from the phosphate as insoluble aluminum phosphate precipitate from the solution equilibrium, this fluorescence is extinguished. Shortly before the equivalence point is reached, there is a linear relationship between the fluorescence and the residual aluminum content, which can be used in a quantitative intensity measurement for extrapolation calculations (principle of standard subtraction). Alternatively, the end point of this analytical precipitation titration can also be indicated by means of known adsorption indicators, which are able to show the end point more sensitively.

In embodiment 6, the determination of a heavy metal is described by a complexometric titration according to the invention.

To determine cadmium, appropriately immobilized EDTA is used as the titrator. The titer per micro-reactor must be at a sample volume of 0.1 ml are in the nano equivalent range if samples are to be measured in the ppm range. The buffer that is also to be immobilized should be at pH> 9. The reagent cocktail is used as an indicator, which is also used in the dry chemical colorimetric test strip method. Other selective indicators for cadmium are mentioned in the literature mentioned above.

For the determination of fluoride according to embodiment 6, the intensely red-colored iron (III) rhodanide serves as the titrator, which is decolorized in the course of the two-dimensional titration by the formation of a strong iron-fluorine complex. The titrator is therefore self-indicating. If the fluoride is to be measured in the ppm range, sample volumes of 0.1 ml and titrator concentrations in the nanomole range are required per micro-reactor.

Embodiment 7 describes the determination of an antibody / antigen partner by means of a micro-channel column

This embodiment corresponds to a precipitation or complexometric titration. In the quantitative determination of an antibody, the antigen immobilized on a surface serves as a titrator. It is present in the microreaction chambers or channels as an associate with a colored antibody with a lower affinity constant than the analyte.

It can thus be released by the latter and will subsequently diffuse away, as a result of which the coloration of the chamber is extinguished. The diffusion can be accelerated using capillary suction forces. For the sake of sensitivity, it becomes a marker HS preferably uses a fluorescent compound.

An antigen as an analyte is determined analogously with the associated antibody as a titrator. The latter is saturated with a labeled antibody which has a lower affinity constant than the analyte to be detected.

Example 8 deals with the implementation of immunoassays with enzyme labeling by means of an ELISA method.

In this exemplary embodiment, an immunoreactor module is connected downstream of the sampling module. In the

Micro-reactors of this immunoreactor module contain the analyte in enzyme-labeled form bound to its partner. The latter itself is covalently attached to a surface. After opening the connection channels between the two modules, the more strongly bound analyte displaces its enzyme-labeled derivative from the binding sites, which makes the enzyme labeling mobile and can be conveyed into the quantification module with a small amount of liquid. There it reacts for a certain time with the corresponding one

Substrate before the substances generated or consumed in the process are precisely recorded in the titration module. When using glucose oxidase as the labeling enzyme, the titration module can be constructed analogously to the module for measuring hydrogen peroxide already described.

In embodiment 9, the determination of the chemical oxygen demand (COD value) and the TOC value is sketched using a device according to the invention. SO graces.

An embodiment of an acid and heat-resistant sample preparation module loaded with dichromate can replace the extremely environmentally harmful classic COD determination according to DIN and at the same time also provide the TOC value. The individual micro-reactors contain variable amounts of chromate in the micro-molar range. They can be connected by means of a suitable micro connection channel circuit by controlled displacement of the modules against one another and corresponding spacers with micro gas scrubber reactors for measuring the carbon dioxide formed. The micro-scrubbers contain variable amounts of NaOH with the indicator phenolphthalein. After sampling an acidified sample, the aliquots (number corresponding to the desired accuracy) are transferred to the oxidation module. By moving the oxidation module, the connection to the capillary pipettes is interrupted and the connection leading to the individual gas washers is opened. The system is then stored in a drying cabinet at 100 ° C for two hours (DIN). To prevent drying out, water must be offered. For this purpose, either a tub is placed around the sampling capillary, the device is placed in a water bath, or the heating is carried out using steam. During the heating phase, the dichromate oxidizes the oxidizable constituents present in the sample aliquots, including organic compounds, and is itself reduced to a green chromium (III) compound. This happens in all micro-reaction chambers in which the chromate is present in a stoichiometric deficit. Suitable indicators for chromate can also be used to increase the sensitivity SA can be added. The COD value can be read off directly on a scale, which is arranged next to the matrix of the micro-oxidation chambers, in mg 0 ₂ / L. This process reduces the hazardous waste of the DIN process by several orders of magnitude. Simultaneously, however, the micro gas washer can be located, in which the phenolphthalein is becoming colorless. Here the TOC value can be read in absolute units on a separate scale.

For the TOC (Total Organic Carbon) determination, the organic components of the sample are oxidized to carbon dioxide in the oxidation module by strong oxidizing agents, such as dichromate, cerium (IV), hydrogen peroxide plus UV light etc. at a suitably selected pH value and then transferred into the titration module by means of a gas-permeable membrane and "titrated" as analytical hydrogen-carbonate.

Embodiment 10 deals with water determination according to Karl Fiεcher using a solvent-resistant device according to the invention.

The known Karl Fischer reagents are immobilized in variable amounts in micro-reactors under anhydrous conditions. The traces of water penetrating into the chambers and in the liquids to be tested react stoichiometrically with the Karl-Fischer reagent, iodine tetravalent sulfur being oxidized to hexavalent and reduced to iodide. As soon as a stoichiometric excess of elemental iodine is present, all of the water in the sample volumes has reacted. This point is shown more clearly by the use of starch. SZ

Exemplary embodiment 11 describes an absolutely measuring enzyme reactor biosensor for glucose.

Because of the desired freedom from calibration, the entire glucose present in the sample volume taken must be converted to gluconic acid and hydrogen peroxide using the enzyme glucose oxidase (GOD). A correspondingly small amount of blood (in the microliter range) and a sufficient amount of GOD should therefore be used in the enzyme microreactor. Because of the mostly limited amount of sample (a drop of blood), the micro-reaction chamber that is particularly suitable here is the embodiment of the invention, which is based on a micro-channel arrangement. The blood sample is aliquoted via a capillary filling pipette and introduced into the device. From there, the sample flows analogously to FIG. 6 through a zone with immobilized GOD, which can already be located within the micro-reaction channel. In this channel, the amount of hydrogen peroxide produced stoichiometrically from glucose is to be determined quantitatively by "titration" with standard reduction equivalents. A gel with a defined potassium iodide content is preferably used as the reducing agent. Starch can be used as an indicator. The blood sample flows filtered or unfiltered (the GOD-loaded microreactor acts as a filter for the red blood cells) into the titration and measuring channel. There, the hydrogen peroxide formed reacts with the analytical reducing agent to form elemental iodine, which is colored deep black by the known iodine-starch reaction until the total amount of hydrogen peroxide has been converted. A section of the titration channel that is strictly proportional to the concentration is colored black. The one in the blood test Existing glucose concentration can be read off on a printed scale in mg / dl or some other concentration (end of blackening). Alternatively, the hydrogen peroxide can also be oxidized by a standard made of potassium permanganate, the potassium permanganate being decolorized to colorless manganese (II).

Since, for example, potassium iodide and potassium permanganate are primary titer substances, there are generic and absolutely indicative test devices for a large number of substances which can be oxidized selectively with their oxides to form hydrogen peroxide.

Of course, in the exemplary embodiments outlined above, in addition to a standard amount of titration reagent, the micro-reactors must also contain a suitable buffer and stabilization cocktail, but finding their optimal composition is not a problem for a person skilled in the art. Alternatively, in the case of oxidases, the decrease in the oxygen concentration can also be measured by means of a fluorescence signal from a dye which is quenched by oxygen.

For other detection reactions, the reactors can also be loaded with dehydrogenases or phosphates which, during the reaction with the analyte, release substances which are stoichiometric and accessible for measurement analysis, such as hydrogen peroxide, NADH or phenol.

Embodiment 12 describes a quantitative gas dosimeter (passive sample collector) from an invented system configuration according to the invention with gas diffusion collectors.

In this embodiment, a special sampling module with narrow, long diffusion micro-tubes is used. These are in direct contact or via a gas-permeable membrane in contact with a collection phase in a further module in which the gaseous analyte is chemically bound. After a prescribed collection time, the analyte in its new compound form is washed by filling the diffusion tubes with a suitable liquid from the collection phase and the amount of the compound newly formed from the gas is titrimetrically according to the invention by means of a suitable stoichiometric reaction with or without chromatographic separation operations determined with equivalence point display.

For example, an ozone dosimeter can be realized by choosing a measurement analytical reduction equivalent as the collection phase and potassium iodide plus starch as the indicator. The micro-reactor in the titration module, which is colored black, indicates that the collected ozone concentration corresponds to the last neighboring reduction equivalent.

Claims

claims

1. Method for the concentration determination of an analyte, analyte mixture or a biochemical analyte-equivalent substance (titrant) contained in a sample with the aid of at least one selective reagent (titrator),

characterized,

that with a known sample volume and known chemical titrator equivalent, a series of spatially separated and quantitatively quantitative titrator-titrant reactions are carried out, the individual titrator-titrant reactions being at least one of the distinguish between two sizes of sample volume and chemical titrator equivalent.

2. A method for determining the concentration according to claim 1, characterized in that the individual titrator-titrant reactions of the reaction series differ at least in that the titrator equivalent is changed incrementally with a constant sample volume or that with a constant titrator Equivalent to changing the sample volume incrementally.

3. A method for determining the concentration according to claim 1, characterized in that the individual titrator-titrant reactions of the reaction series differ at least in that sample volume and titrator equivalent are changed incrementally in opposite directions.

4. A method for determining the concentration according to one of claims 2 or 3, characterized in that the titrator equivalent and / or sample volume are changed incrementally between spatially adjacent Titra¬ tor-Titrand reactions.

5. A method for determining the concentration according to one of the preceding claims, characterized in that sample volumes in the milli- to nanoliter range and milli- to nano-equivalents of the titra- tor are used.

6. A method for determining the concentration according to one of the preceding claims, characterized in that the result of the concentration determination is visual, electrochemical or optical

Evaluation devices such as barcode readers or projection devices are each determined in the course of determining titrator-titrand reactions with excess titrator or titrator deficiency.

7. A method for determining the concentration according to one of the preceding claims, characterized in that in addition, in the titrator-titrant reaction

Indicator reagents and / or auxiliary substances such as buffers, masking or complexing substances, stabilizers or reaction accelerators are used.

8. A method for determining the concentration according to one of the preceding claims, characterized in that after the sampling and before the titrator-titrand reaction, neutralization, complexation, oxidation, reduction, separation or gas washing steps and / or chemical and / or biochemical analyte recognition reactions and / or chromatographic or electrophoretic separation operations which increase sensitivity and selectivity.

9. Device for determining the concentration of an analyte or analyte mixture contained in a sample (titrant) with the aid of a selective reagent (titrator), characterized in that the device comprises a sampling module with a plurality of sampling devices and a titration module with a plurality of spaces ¬ Lich separate reaction areas and that the individual sampling devices are loaded with different sample volumes and / or in the area of the individual reaction areas different chemical titrator

Equivalents are immobilized, the respective sample volumes and respective titrator equivalents being known.

10. The device according to claim 9, characterized gekenn¬ characterized in that the reaction areas are arranged in the order of systematically increasing titrator equivalents.

11. The device according to claim 9 or 10, characterized in that the arrangement of the sampling devices in the sampling module is correlated with the position of the reaction areas.

12. Device according to one of claims 9 to 11, characterized in that the reaction areas in the titration module as depressions with the same or different

Capacity are trained.

13. The apparatus according to claim 12, characterized gekenn¬ characterized in that the depressions have capacities in the micro and nanoliter range.

14. Device according to one of claims 9 to 11, characterized in that the reaction areas on the Titrationsmo¬ module are defined by thick film techniques such as screen printing.

15. Device according to one of claims 9 to 14, characterized in that the reaction areas are arranged in a one-, two- or three-dimensional matrix or in a circular, spiral or helical shape on or in the titration module.

16. Device according to one of claims 9 to 15, characterized in that the individual sampling devices as cavities such as pipettes or capillaries with the same, different or variable holder assets are trained.

17. The device according to one of claims 9 to 16, characterized in that the sampling devices as diffusion

Tubes are formed which are connected to a module containing a collection phase for gaseous analytes.

18. Device according to one of claims 9 to 17, characterized in that the sampling module and the titration module form a unit and the reaction areas are formed within the sampling devices.

19. Device according to one of claims 9 to 18, characterized in that the sampling devices have a closure on the side facing the sample.

20. The apparatus according to claim 19, characterized gekenn¬ characterized in that the closure is designed as a check valve, as a mechanical or micromechanical valve or as a self-dissolving layer.

21. Device according to one of claims 9 to 20, characterized in that between the sampling module and Titrationsmo¬ module a preparation module for oxidation or reduction / Gaεaustreibung and / or electrophoretic or chromatographic separation and / - or for chemical or biochemical analyte bC

Detection, amplification or separation is arranged.

22. The apparatus according to claim 21, characterized in that the preparation module is designed as a permeable membrane, filter, chromatography column or as a spacer having flow channels or diversions.

23. Device according to one of claims 9 to 22, characterized in that the sampling module and / or the titra¬ tion module and / or the preparation module by means of thick film techniques such as screen printing, by means of

Injection molding, CNC technology, is structured or microstructured by means of the LIGA process or by means of semiconductor structuring processes such as photolithography.

24. Device according to one of claims 9 to 23, characterized in that the individual functional modules are designed in such a way that the introduction of the sample volume into the preparation module and / or into the

Titration module by mutual displacement of the modules and / or an intermediate spacer with corresponding recesses and / or by lifting or pushing a piston can be realized.

25. Device according to one of claims 9 to 24, characterized in that spacers, sliding layers or separating layers on or between the individual devices. U

foils are arranged.

26. Method for determining the concentration of an analyte, analyte mixture or a biochemical analyte-equivalent substance (titrant) contained in a sample with the aid of at least one selective reagent (titrator), characterized in that a known sample volume with at least one, is reacted with a known chemical equivalent density of the immobilized titrator and that the titrand concentration is determined by measuring the geometric dimensions of that sub-area of the immobilized titrator in which a specific titrator-titrand reaction taking place quantitatively and analytically took place.

27. A method for determining the concentration according to claim 26, characterized in that a length or area measurement of the Titra¬ tor-Titrand reaction is carried out visually, electrochemically or by means of optical evaluation methods such as barcode readers or analogously to CD players becomes.

28. Method for determining the concentration according to one of claims 26 or 27, characterized in that the sample volume is subjected to oxidation or reduction / gas expulsion and / or electrophoretic or chromatographic separation and / or a chemical or biochemical analyte - Identification, amplification or separation under 1.1 is drawn.

29. Device for carrying out a method according to one of claims 26 to 28, characterized in that at least one titrator channel is arranged on or in a carrier and that the at least one titrator in the region of this at least one titrator Channel immobilized.

30. The device according to claim 29, characterized in that the at least one titrator channel is a linear, circular or spiral channel.

31. The device according to claim 29 or 30, characterized in that the at least one titrator channel has a variable cross-section.

32. Device according to one of claims 29 to 31, characterized in that the at least one titrator channel is a capillary channel.

33. Device according to one of claims 29 to 32, characterized in that the at least one titrator channel has a length of 1 to 20 cm.

34. Device according to one of claims 29 to 33, characterized in that the at least one titrator channel has a diameter in the μm range.

35. Device for determining the concentration according to one of claims 29 to 34, characterized in that the at least one titrator channel is preceded by a preparation channel for carrying out method steps according to claim 28.

36. Device for determining the concentration according to one of claims 29 to 35, characterized in that a sample channel for placing and transporting the sample is in contact with the at least one titrator channel.

37. Device for determining the concentration according to one of claims 29 to 36, characterized in that a cover is provided and the at least one titrator channel is arranged between the support and this cover.

38. Device according to one of claims 29 to 37, characterized in that the sample channel is formed in the cover.

39. Device according to one of claims 29 to 38, characterized in that the sample channel is a capillary channel.

40. Device according to one of claims 29 to 39, characterized in that that a plurality of the same or at least partially different titrators are immobilized in the area of separate titrator channels and the individual titrator channels are arranged next to or in succession.

41. Device according to one of claims 29 to 40, characterized in that the carrier by means of thick film techniques such as

Screen printing, structured or microstructured using injection molding, CNC technology, using the LIGA process or using semiconductor structuring processes such as photolithography.