WO1996035121A1

WO1996035121A1 - Sampling device with integrated dosimeter and function display and sampling process

Info

Publication number: WO1996035121A1
Application number: PCT/DE1996/000821
Authority: WO
Inventors: Karl Cammann
Original assignee: Karl Cammann
Priority date: 1995-05-03
Filing date: 1996-05-03
Publication date: 1996-11-07
Also published as: DE19516179C1

Abstract

The invention relates to a sampling device and a process for the selective collection, enrichment and separation of the materials and/or biological systems (analytes) to be determined from the sample matrix concerned, which permits not only the current functional indication of the state of charge but also a quantification of the analyte on dosimeter-like principles. This is achieved by the invention by specially analyte-selective collection phases on a receptor basis, the analyte binding sites of which are, however, saturated before every use with an easily detected 'indicator substance'. The latter is forced out of the binding sites of the receptor by the analytes to be detected and this can be immediately recognised by the naked eye if the indicator compounds are labelled with bright colours or fluorescence, and in other cases easily detected by means of internal or external sensors. This novel combination of a sampling device and dosimeter has considerable advantages over the prior art devices owing to the recognition of analyte ruptures which become obvious especially in trace analyses or the analysis for extremely dangerous substances.

Description

Sampling device with integrated dosimeter and function display and sampling method

The invention is directed to a sampling device and a method for collecting, enriching and separating the substances to be determined or complex biological and non-biological systems (= analytes) from the sample matrix, which simultaneously perform a functional test in a novel manner combined with a quantification of the substance (s) to be determined (analytes).

The demand for quantitative analyzes of complex mixtures of substances has risen sharply in the fields of environmental analysis and medical diagnostics due to increased environmental awareness and great interest in quick and reliable medical diagnostics and preventive care. In the field of environmental analysis, but also in the field of biological and medical analysis, the quality of a chemical analysis is crucially dependent on the quality the sampling and sample preparation in the laboratory. Especially in the field of trace analysis, the analytes, depending on the detection limit of the analytical method used in each case, must in many cases be enriched or concentrated before the actual chemical analytical determination. In this process, however, the components of a sample (= sample matrix) that are not to be determined (to be analyzed) should not be enriched, since under certain circumstances they can then determine the actual chemical analysis of the analytes because the selectivity of the determination method in question is no longer sufficient can disturb. Therefore, a simple concentration by, for example, evaporating (rotating in a rotary evaporator) the solvent or dissolving the entire gas sample in a small amount of solvent (in the sense of a gas scrubbing) is not the optimal solution.

It is optimal if the enrichment with a

Separation of unwanted sample components is connected. Various sampling techniques have therefore proven successful for chemical analysis of gaseous or liquid samples.

In the case of gaseous samples, an attempt is made to enrich the analytes in conjunction with a simultaneous separation from the main gas quantity by passing the sample gas (as described in many DIN methods) through collecting tubes filled with a special absorption or adsorbent, that the analyte with or without a chemical change (capture reaction) preferably retains and the sample matrix can pass without quantitative absorption or adsorption or chromatographic delay. to In the actual chemical analysis, the analyte molecules so concentrated and more or less freed from the matrix are flushed down from the relevant collection phase by means of suitable measures, for which purpose minimal amounts of gas or liquid are to be used for this elution.

For this purpose, the known gas collecting tubes are, similar to a filled chromatography column, filled with a so-called stationary phase, which binds the substances (analytes) to be determined particularly effectively. For this purpose, according to the current state of the art, adsorption (basis: adsorption isotherms) or absorption forces (basis: Nernstian distribution theorem) are preferably used. Both

Unfortunately, effects are not specific to the molecule and therefore analyte, but act preferentially via the known weak interaction forces (van der Waals, dipole-dipole, hydrophobic interactions, etc.), which e.g. are determined by the polarity of a molecule.

In the case of gaseous samples, the known stationary phases of gas chromatography are preferably used, which have a particularly pronounced binding capacity for the analytes, while the sample matrix components are only weakly bound. A more or less analyte-selective binding and thus quantitative separation from the matrix unfortunately does not occur due to these adsorption or absorption forces (between a gas phase and a thin liquid film - analogous to the relevant chromatography types). According to the prior art, this collecting tube or containers (wash bottles, I pinger or the like) are either good adsorbing substances such as activated carbon, silica gel, aluminum oxide, Mg silicates (Florasil), zeolites, molecular sieves or the like are used. The analytes are then desorbed for subsequent analysis, preferably at elevated temperature (thermal desorption). Eluent can be a gas or a liquid with good solubility for the analyte molecules.

Distribution equilibria are preferably used for liquid samples. Therefore, the corresponding typical stationary phases from the distribution chromatography (compare: author collective (1981), analytics, VEB German publisher for the basic material industry, Leipzig), such as e.g. Carbowax 300, Squalan, Apizon, SE 30, SE 52, OV 17 or preferably the high polymer Tenax and others. used, which are drawn up as a thin liquid film on an inert, granular carrier material. The latter is in the collecting device in question as a collecting phase.

The selection of the liquid film best suited for the analyte and the selection of the suitable eluent depend on the so-called chromatographic triangle, which is familiar to the person skilled in the art. Here, the selection of the stationary collection phase is first made on the basis of maximum interaction forces (polar analytes require polar collection phase, non-polar correspondingly non-polar phases).

Instead of extraction with suitable solvents, which subsequently have to be concentrated and disposed of again for the purpose of concentration, the so-called solid phase extraction (solid phase Extraction, SPE) on solid, stationary phases, which are borrowed from liquid chromatography, for enrichment and retention of the analytes and a matrix separation have proven very successful. In the corresponding collection of analytes from liquid samples, the most successful technique of so-called solid phase extraction is based on a distribution of a preferably lipophilic analyte in a corresponding phase of low polarity. So-called, for example, reversed-phase (RP) filling materials (for example RP-12 or RP-18) are particularly suitable for collecting and separating less polar analyte molecules.

Both the sampling of gases and that of liquid samples can, however, according to the invention in

Design based on the affinity chromatography particularly advantageous. The idea of the present invention is that analyte-specific or at least selective receptors (biological or non-biological type or origin) are used as the analyte collection phase. All substances which have substance-recognizing properties analogous to the principle of the antigen (= analyte) -antibody interaction (key-lock principle or "induced-fit" glove principle) are suitable as selective analyte-binding receptors. At the moment, these are further suitable and also known:

Antigen-antibody-Ab fragments, DNA-complementary DNA (principle of the DNA probes) or analog systems based on RNA, guest-host molecules of the so-called supramolecular chemistry, or stationary carrier materials, in the surface of which the analyte or analytes to be collected - Molecular forms were molded in according to the so-called "molecular imprinting technique", so that the form complementary to the analyte molecular form there as Depressions (impression) occur.

Because of the extremely high specificity and very strong and selective binding of analyte molecules (eg affinity constant approx.> 10 ¹⁰ ), the use of analyte-molecule-specific receptors as the collection phase allows much better analyte isolations and enrichments than all other known ones Method.

When collecting from gaseous samples, however, a certain amount of moisture must be present when using biological receptors (antibodies, antibody fragments, DNA, RNA or the like) so that the protein molecules (tertiary and quaternary structure) function properly. for selective analyte connection is retained; Therefore, in such cases, gas scrubbing with an aqueous or partially aqueous buffer solution is required. In the case of liquid samples, only a certain pH value and a certain ionic strength need be maintained so that the protein structure is not denauturized and the specific analyte binding property is lost.

In all cases, i.e. Both in the case of the first-mentioned collecting materials based on adsorption or absorption (distribution) and also in the case of collecting materials based on analyte-receptor, however, the current loading state (degree of saturation with analyte molecules) of the relevant collecting device and thus also the so-called Analyte breakthrough volume

(loss-causing, uncontrolled exit from the relevant collection device due to overload) of the sample thus collected initially unknown. To determine this sample volume, the maximum amount of analyte retained by the collection device must be used Determine individually for each collection device. This is usually done empirically from experiments with synthetic standards. The person skilled in the art thus receives analyte concentration-dependent, maximum sample volumes at which the analyte or the analytes are still retained 100% in the collection and separation device.

In terms of correct trace analysis, it is of central importance that the recovery, enrichment and matrix separation of the analyte or analytes have a recovery rate of 100%. The breakthrough volume characterizes the amount of sample (volume of sample gas or liquid) which may be passed through the collecting device in a predetermined time before the first analyte molecules leave the collecting device and are therefore no longer available for later quantification . This leads to minor findings.

The breakthrough volume is also dependent on the sample matrix. If there are more molecules of a similar construction to the analyte molecule (or ion) in the sample matrix, the breakthrough volume is significantly reduced, for example, because the collection phase has only a certain, finite capacity. In the case of liquid samples, surface-active substances in the matrix can have a similar effect. Heating also reduces the breakthrough volume. Because of this strong dependence on the individual matrix of the sample in question and the collection conditions, experienced specialists usually take special precautionary measures to ensure a breakthrough (= exit from the collection device while the collection phase is still in progress) To prevent or exclude analytes.

To prevent a so-called overloading of the collecting phase in the respective collecting device and thus a breakthrough of the analyte or analytes and thus a loss, at least one second gas or liquid collecting tube is connected behind the first and also determined in this and all others collection devices connected in series in this way and the analyte (s) and adds these contents to the value obtained with the main collection system.

After the collection and separation phase with enrichment of the analyte or analytes in the collection device (wash bottles, tubes, cartridges, capillaries or the like), in the case of gaseous samples, either the analyte or analytes is removed from the collection by increasing the temperature (thermodesorption) or by extraction with a little solvent. Device brought and chemically analyzed a measured amount. In the solid-phase extraction of liquid samples, the solvent which elutes the analyte best is passed through the collecting device and the analyte (s) is thus fed to the actual analysis. In both cases, of course, the total amount of sample that has flowed through the collecting device is known.

Since both the gas sampling method mentioned above and the one for liquids, any overload leads to faulty analyzes, the problem is to recognize the latter in order to be sure. In the case of strongly fluctuating analyte concentrations and a varying matrix, temperature and sample collection speed, even the series connection of several collection devices may no longer be sufficient. A major disadvantage of the last-mentioned, previously practiced method is the correspondingly multiplied workload and the strong increase in resistance for the sample flow through several devices connected in series. Since the actual collection zone is relatively densely packed (poured) for reasons of short diffusion paths of the analytes to corresponding binding sites on the surface of the mostly powdery collection material, this zone acts as a strong flow obstacle, ie it can, depending on Grain size of the filling, carrier and collecting material, build up considerable pressure losses.

However, since the sampling pump is always placed behind the collection devices (suction of the sample) for reasons of exclusion of impurities, the limits of the sample throughput can easily be reached. One is, for example, that for a reasonable sample speed such a high vacuum must be created behind the collecting device that the sample liquid in question begins to boil. In the protein-chemical collection phases based on analyte-specific biological receptors with corresponding analyte binding sites, the problem of protein denaturation can be caused by a wide variety of causes (temperature, pressure, pH, ionic strength, chaotropic matrix, heavy metals or the like, poisons, etc.) occur and lead to the failure of the collecting device without any other external signs. A denatured protein has lost all antigen (= analyte) binding properties, ie there is an available collection capacity of zero. The relevant collection device is therefore for sampling become unsuitable.

It is also an object of the present invention to provide an improved sampling and collecting device and a method to be carried out with it, which is capable of detecting the current analyte loading (occupancy) state and a simple, automatic functional test of a collecting device for gaseous and liquid samples, whereby, in the sense of a quality-enhancing sampling technique, a pre-sample-like analyte quantification can also be carried out.

The object is achieved according to the invention in that the stationary collection phase in question, which is optimal for the analyte or analytes, is not - as usual - unoccupied, that is to say with free ad or absorption sites, but according to the invention already with a more or less adsorbed or absorbed or receptor bound compound (here: displacement substance or indication compound or substance called) is used (saturated with this substance to be displaced) is used. The displacement substance / indication compound which can be easily replaced by the analytes to be collected is suitably marked according to the invention, so that a small, downstream, responding to the marked substance responding to the marked substance displaced by the analyte or analytes is used by means of a marking zone or an integral signal which is as easy to recognize as possible ¬ flow detector (or internal and / or external sensors can easily determine whether the analytes to be collected have already completely displaced this marking substance or this indicator compound) from the active collecting phase area of the device or not, which would be a breakthrough to avoid with the known disadvantages. The integral signal represents the summation of all detector (sensor) signals and corresponds to the known total amount of indication compound that has been displaced by the analytes up to the point in time. Since the total capacity of the collection phase at analyte binding sites is known, any remaining collection capacity can be determined in this way.

The indication compound to be displaced by the analyte usually exists. from an analyte-like binding molecule with poor binding to the collection phase as the analyte molecule or molecules, but which is additionally covalently provided with a suitable label or contains one intrinsically.

The type of marking of this indication substance is irrelevant according to the invention. However, those which are particularly advantageous are those which are visible to the naked eye and / or minimal aids, e.g. can be determined by means of simple internal or external sensors.

The selection of the optimally suitable labeled compound, which is to be displaced by the analyte molecules in the course of sampling, is carried out in the case of the chromatographic collecting materials on the basis of the chromatographic triangle, in that a basic substance is used as the molecule to be displaced, which is given under the given conditions are hardly or very poorly retained on a corresponding adsorption or distribution chromatographic column. If, for example, the analyte molecule type to be enriched and collected is a particularly lipophilic compound, which preferably has an RP phase are retained, the labeled displacement substance must be less lipophilic, which can be expressed by a distribution coefficient that differs by a factor of> 10. In this case the surface of the collecting phase is also lipophilic (eg RP-18 phase). If, for example, the analyte is a hydrophilic, polar compound, a polar collecting phase surface is preferably chosen for the enrichment and separation of the analyte, and a less polar marking substance is used together with a likewise less polar solvent.

In the novel collecting materials based on analyte receptors (for example immunochemical binding by means of antibodies or analyte-binding antibody fragments and molecular imprinting technology), the analyte molecule itself is suitable according to the invention if it is, for example, covalently attached to larger marker atom groups (conjugated group of molecules with suitable optical, electrochemical or other properties suitable for labeling). In this case, the steric hindrance of the analyte molecule labeled in this way leads to a smaller affinity constant, which corresponds to the less rigid connection (or association) intended according to the invention to the receptor phase.

Suitable marker molecules or groupings of atoms have proven particularly useful in many of our own test runs: stable dye molecules with particularly high molecular extinction coefficients, fluorescent molecules with strong emission intensity in the visible range of the spectrum, redox systems based of Molecules or ions with a particularly high standard exchange current density, furthermore: electrochemically active (easily reducible or oxidizable molecules without high overvoltage) as well as certain ionophores, enzymes, mass-increasing groups, DNA / RNA labels or the like, as described by the immunoassays or DNA probes are known.

In the case of labeling groups which are too small in molecular terms and which, when covalently linked to the analyte molecule

To lower the affinity constant too little, so that this indicator compound is displaced too poorly by the analytes, it is also possible according to the invention to take a similar analyte molecule with an epitope suitable for the receptor molecule in question, if this labeled compound then has a sufficiently weaker bond with the binding site of the relevant one Re¬ received. Sufficiently according to the invention means an affinity constant which is at least 2 times smaller.

In the case of the collecting materials based on analyte receptors, the type of inert carrier material is irrelevant for the analyte-recognizing receptor molecules. It is only important that the carrier material in question does not lead to an excessively high pressure loss in the mostly column-like collecting devices.

According to the invention, the capacity of the collecting space can also be increased by a cascade arrangement of filter papers loaded with corresponding analyte-catching receptor molecules or sample-permeable plastic films with correspondingly larger diameters. State of the art in the field of inert carrier materials for biological and non-biological see receptors are porous, pressure-stable supports (eg controlled pore glass, CPG, plastic particles or those based on inorganic materials, such as silica gel or the like).

Significantly lower pressure losses occur in a sample collection device filled according to the invention with a rolled-up planar carrier (e.g. filter paper, dialysis foils, cellophane or plastic membranes).

Here, the analyte-selective receptor molecules can be immobilized on both sides of the carrier, which operates through spacing devices with defined gaps. The person skilled in the art knows corresponding arrangements from the field of desalination plants based on reverse osmosis or electrodialysis. The resulting short diffusion distances of the analyte molecules to the relevant binding sites of the receptors greatly accelerate the kinetics of the binding of the analyte molecules to the relevant binding sites.

Furthermore, dialysing tube-like arrangements (for example, originating from the blood wash in kidney patients) that have been converted as a collecting device can be used according to the invention if the surfaces that come into contact with the sample are coated with immobilized, analyte-binding receptors. Such an arrangement can, for example, contain well over 100 receptor-loaded tubes with individual diameters of less than one millimeter and lengths of more than 10 cm. The test flow can combine both through the hoses and around the hoses as well as when suitable flow characteristics are present respectively. A longitudinal flow is important. Such an advantageous arrangement of the collecting phase achieves a large loading capacity with a very low pressure drop. Advantageous embodiments consist of transparent materials and allow the wavelengths necessary for simple optical detection to pass through, which enables the loading state to be easily recognized by the naked eye. The respective other surface of the tubing, which is not in contact with the sample, can, if necessary, also be rinsed with a solution of higher osmolarity when using dialysis tubing, so that the analyte molecules to be collected also get through a flow of the solvent molecules is driven close to the binding sites of the receptors. If low-molecular analytes have to be separated from high-molecular ones, then the analyte-binding receptors are advantageously immobilized on that side of the dialysis membrane which is not in contact with the sample, because in this way a double-acting separation is achieved with a correctly selected molecular cut-off becomes. This sampling technique is particularly suitable for obtaining analytes from biological or medical samples.

The correct orientation of the analyzer-recognizing receptor molecules is that in which the specific analyte binding site points away from the inert support surface, since rapid and unimpeded binding of the analyte to be collected then occurs and thus also an optimal binding site density is given is. For this purpose, numerous methods are described in the literature which are not the subject of the invention and are familiar to the person skilled in the art. In the method according to the invention, the receptor-assigned collection and collector phases for the analyte molecules in question are saturated with the indicator compound before each new use of the analyte collection and separation device (loading to avoid excess). The loading process can be determined either directly or indirectly (eg irradiation with light of a suitable wavelength in the case of fluorescent markers), visible coloring of the collecting zone or in the case of opaque collecting devices and / or other marking by the breakthrough of this indication connection in the flow detector. The washing out of an excess can also be tracked by the detector.

Since the indication substances marked in this way are gradually displaced from the collection zone in question in the course of sampling by the differently behaving analyte molecules, the invention thus provides a multiple and immediate possibility of checking the loading capacity of the sampling or loading capacity that is still present. Collecting device. This solves an existing problem of trace analysis of complex samples particularly effectively.

With a constant and reproducible packing density of the active collecting zone, according to the invention there is preferably also a quantitative one with the particularly selective acting collecting materials based on the receptor by simply measuring the marking zone (distance) changed by the analyte molecules to be collected after corresponding calibration Note that can serve as a preliminary test. This dosimeter method can also advantageously be used to determine the optimum analyte concentration for the subsequent analytical determination. be attracted, which saves unnecessary additional regulations. At the same time, the reliability of the analysis in question is increased by the redundancy of the results.

In the case of the use of suitable receptors which selectively bind certain classes of substances, which is possible according to the invention, a new and particularly simple and fast method for the reliable determination of a large number of corresponding sum parameters results by means of such a new collecting device. It goes without saying for a person skilled in the art that such a collecting device, which according to the invention is combined with a status display which also serves as a dosimeter, can be calibrated in a wide variety of units, such as, for example, mass or molarity.

The present invention thereby combines the known techniques of analyte enrichment and matrix separation in gaseous and liquid samples with a new technique of quantitative test tube analysis based on analyte receptor.

In the case of a collection mechanism based on chromatography, judging from the current state of the art, no indication substance is used for on-line visual control of the mode of operation, which would also allow quantitative estimates of the analyte concentration. All of the sample collection devices previously described in the literature propose that such devices be connected in series to avoid analyte losses. All standardized processes (eg DIN, EPA and other regulations) express themselves similarly. The prior art in the field of test tubes or dosimeters has hitherto been characterized in that more or less selective chemical reactions of the analyte with certain reagents take place, but which as a rule lead to an irreversible change in the

Lead analytes. As a result, such devices are less suitable for sampling, sample enrichment and separation of analytes. Dosimeters based on this have been in use for a long time and can usually be do not regenerate for further measurements.

In the case of the analyzer-receptor-based sampling device according to the invention, the analyte molecules are bound and associated unchanged and reversibly to the receptor molecules. They can then be split off again unchanged by known processes which are gentle on the receptor molecule (change in pH value, chaotropic reagents) and can be subjected to a chemical analysis. Characteristic of the present invention is therefore also the reversibility of the collection, enrichment and separation mechanisms as well as the regenerability of the dosimeters thereby given.

Direct on-line immunochemical monitors based on the displacement of a weakly bound fluorescence-labeled antigen from an affinity-chromatographic column in the form of a corresponding cartridge are known. Here, however, the registration of the fluorescent compound emerging from the cartridge serves as a direct analysis signal (determination signal). In no case is the quantification carried out by measuring a stretch of the collecting phase. In the assay methods known to the person skilled in the art, the optical detector signal the "immuno-reactor" tends towards zero, which is difficult to distinguish from the state with low analyte traces. In contrast, in such a case in the present invention the marked zone in the collecting device remains almost unchanged. However, the current load status can still be read immediately.

Further direct determination techniques in miniaturized kit form are also known, in which a marked reagent antigen diffuses into a closely adjacent reaction space after corresponding displacement by the analyte antigen and there, for example with the aid of additional reagents generates a sensitive chemiluminescence that is measured directly. These and all other methods for the direct display and quantification of small molecules are direct determination methods on an immunochemical basis (in unoassays) and no sampling techniques or a combination of sampling and dosimeter techniques.

The present invention primarily represents an optimized and particularly advantageous sample preparation technique which combines the collection of the analytes of interest with their enrichment and separation from the sample matrix and which the actual highly precise determination of the analyte or analytes by any means Methods allowed. According to the invention, the type of analyte binding by very selective immunochemical bonds or more physical-chemical adsorption or distribution is irrelevant. What is important according to the invention is the simplicity of the marking and its detection and thus the reliable display of the (still) present Analyte collection capacity, which in many cases can also serve as a dosimeter at the same time.

Because of the large number of possible combinations of stationary collection phases with the most diverse flowing phases, the invention enables a not obvious extension of all the immunoassay techniques described so far. The invention therefore relates to an ideal combination of sampling techniques with enrichment and separation techniques, wherein depending on the selectivity of the collection phase selected beforehand, only a single analyte or also a certain class of substance (determination of the sum parameters) can be collected.

The analyte thus automatically enriched and separated from the often disruptive sample matrix is obtained quantitatively according to the known solid phase extraction methods, the optical tracking of the washing out of the rest of the indication compound allowing the extraction effectiveness to be controlled; if it is not extracted, the collected analyte molecules will certainly not. In immunochemical methods (affinity-controlled collection of one or more types of analyte molecules), the known techniques of gentle antibody-antigen dissociation, such as elution using chaotropic reagents (eg urea, etc.) or an acidic buffer solution (pH between 1 to 3) used for this.

The amount of eluent is dimensioned such that the collection phase is then freed of the labeled compound ^'.

Have extensive tests prior to the invention 21 show that well (correct orientation) immobilized affinity phases survive many of these regeneration steps without a significant loss in functionality. They can be reloaded with the marked antigen to be displaced by reloading under optimal binding conditions (pH value, ionic strength, etc.) and used for the next sampling. Even with this loading with the indication compound (marked antigen), an easily visible marking is of particular advantage, because the loading state can be recognized immediately, and therefore the point in time when no excess marking substance leaves the collecting zone during the rinsing process.

The free choice of an optimal analysis method for the analyte separated and enriched according to the invention results in particularly advantageous degrees of freedom with respect to the decisive analysis and determination method, which in addition to the separation of disruptive additions to the particularly reliable and correct ones Lead analytical findings.

Use of this self-controlling analyte collection device thereby improves the detection limits and correctness of trace analyzes in gaseous and liquid samples, while at the same time simplifying and saving time. As a result, the invention represents a decisive further development of all known bio- or immunoassays based on a displacement reaction, which in contrast do not have any freedom from determination methods.

Accordingly, the invention solves several problems trace analysis of gaseous and liquid samples which cannot be solved by the known immunoassays and test tube techniques alone.

The present invention achieves the following advantages, which are extremely important particularly for trace analysis: a) The current loading state of the relevant collecting device can be recognized immediately, i.e. New and fresh can be differentiated from the state old, used or still loaded with analyte or saturated with other substances. b) It can be seen whether in the sample matrix the substance to be analyzed is worth mentioning

Quantities are present; if the marking zone does not change during the passage of the sample, there is nothing to be analyzed later, which saves analysis time, costs and chemical waste. In addition, an optimal amount of sample can be drawn almost automatically and in situ and without prior information about expected analyte concentrations. c) After a suitable calibration, the amount collected up to that point can be determined from the section in which the indicator substance has been displaced by the analyte (test tube principle). d) It can be determined immediately if the capacity of a sampling device is nearing its end or has already been exceeded, which prevents analysis errors. e) No further sampling devices are required in series in order to prevent the burnout. prevent what keeps the pressure loss within limits and saves working time for the separate refurbishment. f) The collecting and separating devices can be further miniaturized and, for safety reasons, do not require such a long flow path of the collecting phase, but can, because of the possibility of checking, work with enlarged, flow-through areas, which leads to less pressure loss and less Pump power required. g) When the analytes collected in this way are obtained by elution of the analytes thus separated from the sample matrix at the same time, the amount of eluent which is necessary for washing out the entire amount of analyte collected without loss is that which is still present Amount of labeled compound displaced from the sampling device. This prevents unnecessary dilution. h) The dimensions of the collecting device and thus also its total collecting capacity can be adapted exactly to the subsequent analytical determination method, ie if an optimal sample quantity for the subsequent determination has been collected by the disappearance of the marked collecting zone, the Collection process is aborted and the precise analyte determination begins, i) the analyte concentration-dependent sampling time avoids unnecessary collection times, which may lead to high analyte concentrations and again require an additional dilution step before the determination, j) with a stoichiometrically constant displacement of more easily bound, appropriately marked In the case of dangerous analytes (eg toxins, viruses, bacteria or the like), receptor partners can dispense with excessive enrichment and subsequent elution if the label represents a DNA / RNA label that is extremely extreme with the known PCR technique is sensitive to determination.

Exemplary embodiments of the invention are explained in FIGS. 1 to 7.

A particularly advantageous, because simple optical version of the invention is shown schematically in FIG. 1. For this purpose, a glass or quartz tube (UV-permeable) 1 with the relevant analyte-optimized collection phase 2 with an inert material 7, receptors 6 and indication substance 5 is used using two fixation elements made of glass or quartz wool or equivalent Fries 3 filled. If intensely colored suitable indication substances or antigen conjugates with particularly large molecular extinction coefficients (such as phorphyrins, carotenoids, crystal violet, etc.) are used, the currently decontaminated capacity in the device can be read off immediately using the decolored zone. After appropriate calibration, the dose can also be calculated.

Colored antigens to be displaced by the analyte molecules are obtained from the analyte molecule itself by covalently linking it chemically with known, stable and intensely colored dyes or chromophoric groups. However, molecules similar to the analyte with a clearly weaker affinity for the enrichment phase can also be used. the. In the case of non-immunochemical analyte binding, the lipophilicity of the labeling molecule (indication substance) can be adjusted by a suitable choice of the chemical property of the chromophoric or fluorophoric group.

For reasons of sensitivity, all known, stable fluorescent labels from the immunoassay or immuno sensor technology are particularly well suited here. The exact type doesn't matter here. For this purpose, the sampling tube 1 must then be illuminated with the relevant known excitation wavelength via a radiation source 4. Fluorescence in the visible range of light is advantageous here, since the analyte-loaded, non-fluorescent zone is then easy and easy to recognize.

Fig. 2 shows an embodiment by means of a detector display 8, which can be used according to the invention when the sample collection device for

It is opaque to light or if an electrochemical label, such as ferrocene, or molecules with a quinoid structure or with other groups that are easy to reduce or oxidize, or with other sensor labels, is coupled to the analyte molecule. To display the loading state and thus the mode of operation, an electrode array covering the entire collecting phase or an additional external flow detector 8 is required either on the inner walls of the collecting tube. Alternatively, of course, a corresponding optical flow detector can also be used at this point for the integral detection of the displaced marker molecules. 3 shows a further exemplary embodiment with a further type of marking. For example, enzymes can also be used as markers for the analyte molecule conjugate compound to be displaced. Here, however, must be in front of the detector

A corresponding substrate, which is converted by the displaced enzyme-labeled compound into a detectable product, is added to the addition device 9. Particularly suitable, since they are already commercially available because of their use in corresponding immunoassays, are analyte markings with, for example, glucose oxidase (GOD) or alkaline peroxidase (POD) or phosphatase, in the presence of which enzymes (displaced from the enrichment) and collecting zone) from the substrates to be added glucose, respectively. Hydrogen peroxide, respectively. p-amine top zoyl phosphate, electrochemically very good to be displayed compounds are known to the average person skilled in the art.

4 shows a schematic embodiment for an optimal in-situ sample preparation for gaseous and liquid samples. In the case of gaseous samples, the gas to be examined is freed from the analytes quantitatively by means of a so-called scrubber 10 (gas scrubber) and the latter is converted into a liquid phase before being placed on the collecting device. This liquid phase can consist of an aqueous buffer solution or - depending on the stability of the receptor molecules - also of an organic solvent. The liquids containing the analytes are then passed through the collecting device, where the analytes are separated and selectively enriched. 27

In the case of liquid samples, a small amount of a buffer solution with a high buffer capacity is advantageously also added to the sample stream by an immunochemical collection device. The pH should correspond to that at which the analyte is optimally bound to the binding site in question.

5 shows an example of a device with a cascade-like arrangement of the collecting phase supports, which can also have a different selectivity for the simultaneous collection of several analytes.

6 shows a further example of a collecting device based on the dialysis tube arrangement with parallel individual tubes, which leads to high loading capacities.

7 shows a biological application in which the analyte or analytes are so dangerous that only minimal amounts thereof are collected and those also remain in a cartridge 12 with a selective collection phase to be placed in front of a syringe 11. Here, the sample is passed over this receptor-loaded phase by suction via the needle 13, which then releases an equivalent amount of the harmless indication substance 14 when the syringe plunger is pushed back for further analysis. In such cases, labeling using DNA / RNA labels is particularly advantageous since the PCR technique can then be used to analyze these traces.

Claims

claims

1. Device for sampling gaseous and liquid samples for the purpose of selective enrichment and matrix separation of substances to be determined and / or biological systems (analytes) with integrated dosimeter display as a function display, that is, a d u r c e g e n e z e i c h n e t; that the current state of loading of analyte- and / or substance group- or biosystem-selective collection phases can be determined by marking with an indication substance, in which the displacement of the indication substance from specific binding sites of the collection phases by the substances to be collected / Biosystems can be used as dosimeters and to check their functions.

2. Device according to claim 1, characterized in that geometric shapes are realized in the marked collecting zone of the device, which enable quantification over a route scale or a counting of indication substance-free zones of the collecting bases (without marking).

3. Apparatus according to claim 1 or 2, characterized in that the analyte-selective and marked collecting phases are immobilized on inert carriers, which as granular bulk material with medium diameter on a micrometer scale in translucent, tubular or hose-shaped or rectangular flow-through Arrangements result in a minimal loss of pressure and that the label-free zone, which is created by displacement of the indication substance by analyte molecules and is clearly visible, enables quick and easy quantification, which can be determined with the naked eye.

4. The device according to claim 3, characterized in that the inert supports for the analyte-selective and marked collection phases are permeable and impermeable foils, which are either cascaded with a large cross-section in series, through which the sample flows or perpendicular to which the sample flows Surface in a rolled-up form, provided with defined spacers, to which the sample is applied, one or both carrier surfaces being covered with the collection phase.

5. Apparatus according to claim 4, d a d u r c h g e k e n n z e i c h n e t that with a cascade arrangement of the inert

Carriers which contain different collecting materials which bind different analytes and thereby form a multi-dosimeter arrangement.

6. The device according to claim 3, d a d u r c h g e k e n n z e i c h n e t that the inert carrier individually or bundled arranged, tubular or tubular membranes or capillaries, wherein the

Flow through the sample on both sides simultaneously or sequentially, i.e. only on the outside and then on the inside through the hoses, tubes, capillaries or vice versa.

7. The device according to claim 6, characterized in that the tubular membranes are dialysis arrangements from the medical field, the collection phases are immobilized on the surfaces of the dialysis tubing by the methods of the straicht¬ th covalent connection and that by an additional osmotic gradient over the dialysis membrane an increased solvent flow perpendicular to the membrane surface can be generated, which supports the analyte attachment kinetics.

8. The device according to claim 1 or 3, characterized in that the marked and selectively analyte-binding collection phases either selectively bind the substance to be determined or selectively remove a substance class to be determined or the biological system to be collected by binding from the sample stream quantitative .

9. The device according to claim 3 or 6, characterized in that as the marked analyte-binding collection phase selectively acting receptors corresponding to the substances to be collected are immobilized in a molecular orientation with accessible binding sites on the carrier surfaces.

10. The device according to claim 9, characterized in that the selectively analyte-binding receptors fol¬ following molecule classes or mixtures of different, belonging to a class or ver¬ different classes belonging receptors include: a) immunological antibodies or antibodies - fragments with the substances or biosystems to be collected as antigen; b) complementary DNA / RNA to that which is to be isolated or bound to be collected (DNA probe principle); c) supra-molecular host-guest connections; d) surfaces obtained by "molecular imprinting" technology, selectively recognizing the complementary analyte molecular form; e) stationary phases of chromatography; in the case of molecule classes a) to c), one not immobilized on the support surface

Partner is collected from the samples.

11. The device according to one of claims 9 and 10, d a d u r c h g e k e n n z e i c h n e t that the receptors with a weaker than the

Indicator-bound (associated) and slightly directly optically (with the naked eye) or by means of simple internal or external sensors displayable indication substance are saturated as a marker compound. 32

12. The device according to one of claims 1 to 11, characterized in that the indication substance contains a formula or the analyte structure or physically-chemically similar molecule which has the property of the substances to be collected easily from the specific binding and association tion with the receptor and can be detected optically or indirectly by means of internal or external sensors.

13. Device according to one of claims 1 to 12, that the indication substance is the analyte molecule or the substances to be collected or biochemical

Systems themselves, modified by a marker atom grouping, contains.

14. Device according to one of claims 1 to 13, characterized in that it has a flow detector, which indicates the current state of the collection device and indicates a total dose via the integral of the flow signal from the marked substance displaced by the analytes .

15. Device according to one of claims 1 to 14, characterized in that the quantification of the indication substances displaced by the substances to be collected is carried out by means of internal or external sensors on an optical, mass-sensitive, calorimetric or electrochemical basis.

16. The device according to one of claims 1 to 15, which also includes a linear sensor array which is parallel to the sample flow and which enables a status display (degree of occupancy) and dose quantification on a connected computer.

17. Method for sampling gaseous and liquid samples for the purpose of selection enrichment and matrix separation of at least one

Analyzes combined with a dosimeter-like quantification. characterized in that analytical and / or substance group or biosystem selective collection phases are loaded with at least one stable and easily detectable indication substance before each sampling, which during the collection process of the substances / biosystems to be collected from the surface of the Collection phase is displaced, the

Analyte dose results from the amount of the analyte-displaced indication substance (label-free zone), which is detected by a distance measurement or by means of an integrated detector signal.

18. The method according to claim 17, characterized in that the analytes are removed from the sampling device by means of an eluent and supplied to a further chemical analysis.

19. The method according to claim 17, characterized in that in the case of dangerous (toxic) analytes, too much enrichment is dispensed with, for which purpose a weakly bound partner of the receptor in question, which is provided with a DNA / RNA label, is chosen as the indication substance to be displaced and the equivalent amount of the indicated indication substance released by the analyte is measured by means of extremely sensitive PCR technology.

20. The method as claimed in claim 17, so that auxiliary reagents are fed into the sample or liquid stream to determine the extent of the marking displacement.

21. The method according to claims 19 or 20, characterized in that the device is positioned in the form of a receptor-filled cartridge on a medical needle or syringe and, after sucking in a certain sample volume, the DNA / RNA displaced in front of the analyte contained therein -labeled indication substance molecules in another container for further PCR-based quantification.