Method for automatic online detection of at least one biological target substance in a sample liquid by means of an online analyzer

Abstract

The invention relates to a method for automatic online detection of at least one biological target substance in a sample liquid by means of an online analyzer (1), comprising: - producing a concentrated sample liquid from at least one first initial volume of the sample liquid by means of: e. dosing and transportation of a first initial volume of the sample liquid via a liquid supply line (25) into a first concentrator chamber (8) of a concentrator unit (3, 33); and f. reduction of the first initial volume of the sample liquid in the concentrator unit (3, 33) and associated concentration of the at least one biological target substance present in the first volume of the sample liquid by means of a first amount of a superabsorbent polymer (24), wherein the reduction and the associated concentration is effected by incubation of a mixture formed from the first initial volume of the sample liquid with the first amount of the superabsorbent polymer (24); - transporting at least one portion of the concentrated sample liquid produced from the concentrator unit (3) into a detection unit (4) of the online analyzer, qualitatively or quantitatively determining the biological target substance present in the concentrated sample liquid in the detection unit (3) by means of a sensor, by means of an immunological detection method, by means of measurement of an amount of adenosine triphosphate present in the sample liquid, by means of a flow cytometry method, by means of a nucleic acid hybridization method and / or by means of measurement of metabolic and / or enzymatic activity of microorganisms. The invention also relates to a device for carrying out the method.

Description

[0001] Method for the automated online detection of at least one biological target substance in a sample liquid using an online analysis device

[0002] The invention relates to a method for the automated online detection of at least one biological target substance by means of molecular genetic methods in a sample liquid, e.g. water or wastewater, a concentrator unit for the automated concentration of at least one biological target substance in a sample liquid, and an online analysis device for the detection of at least one biological target substance by means of molecular genetic methods in a sample liquid.

[0003] The detection of biological parameters plays an increasingly important role in the process industry, for example, in the monitoring and purification of industrial or wastewater, in food production, and in the pharmaceutical and life sciences sectors, for example, in the monitoring of biotechnological processes carried out in fermenters. Target substances to be detected or monitored can include viruses, bacteria, and plasmid-associated bacterial resistance genes.

[0004] The detection of such biological target substances in liquids can be performed using molecular genetic methods, preferably using amplification techniques such as PCR or real-time PCR. For this purpose, samples of the liquid to be analyzed are taken and analyzed in specialized laboratories equipped with the necessary equipment. Manually operated or automatic sampling devices and sample collectors are available for automated sampling from processes, e.g., from clarifiers, fermenters, production vessels, or pipelines. The collected samples are usually transported to a laboratory for further analysis, where the extraction of the sample nucleic acids and the specific and, if necessary, quantitative detection of the biological target substance are carried out manually or at least partially automatically.

[0005] For example, in the field of water and wastewater analysis, online analysis devices are known that perform fully automated sampling and quantitative determination of a (usually inorganic) analyte in the sample. Such online analysis can be performed continuously or discontinuously.

[0006] US 2015 / 0224502 ​​A1 discloses a sample collection device for flow-through sampling in water bodies. The device comprises several flow-through sample cartridges designed to collect water from the water body to be monitored and, if necessary, retain samples of components of the liquid or solids in a filter or adsorption medium, while the collected water is returned from the cartridges to the water body. The samples can either be stored for later laboratory analysis or analyzed directly in the device. For this purpose, for example, biological substances adsorbed on the filter or adsorption medium can be released as a lysate and made available to an analysis module for further analysis, e.g., using qPCR.

[0007] When the concentrations of the target substances in the liquid to be analyzed are low, the problem arises of ensuring that a sufficient amount of the target substance is available during sampling for subsequent analysis. With flow-through sampling as described in US 2015 / 0224502 ​​A1, dissolved substances or solids present in the water even in low concentrations can be retained in the filter medium by passing a sufficiently large volume of water through it. However, this is only practical if the liquid to be analyzed can be returned to the medium without restrictions after the sample has been taken, as is the case, for example, in water body analysis. When monitoring processes, e.g. in water treatment, the food industry or the pharmaceutical sector, it is often not possible to reintroduce the sampled liquid.There is a need for a method for the efficient concentration of sample components to be detected, which makes it possible to keep the volume of liquid to be removed from the process for analysis low.

[0008] A further disadvantage of the device described in US 2015 / 0224502 ​​A1 is that the substance retained in the filter or adsorption medium is passed from the filter or adsorption medium into the analysis module by lysis and elution. On the one hand, it is expected that only a relatively small proportion of the target substance will pass from the filter or adsorption medium into the analysis module. On the other hand, a certain minimum volume of liquid is required to rinse the filter, so that the solution entering the analysis module has a relatively large volume with a correspondingly low concentration of the target substance.

[0009] The object of the invention is to enable improved automated online detection of a biological target substance in a sample fluid and to provide an improved method and device for automated online detection of a biological target substance in a sample fluid. In particular, the method and device are intended to enable efficient concentration of the target substance and low-loss transfer of the sample fluid containing the concentrated target substance to a subsequent detection unit.

[0010] This object is achieved by the method for the automated online detection of at least one biological target substance in a sample liquid by means of an online analysis device according to claim 1, by the concentrator unit for the automated implementation of a concentration of a target substance in a sample liquid according to claim 14 and by the online analysis device for the detection of at least one biological target substance in a sample liquid according to claim 15. Advantageous embodiments are specified in the dependent claims.

[0011] The solution is based on the use of so-called superabsorbents, with which any aqueous sample liquid can be processed in order to concentrate the biomolecules contained in the sample liquid.

[0012] Superabsorbent polymers (SAPs) are plastics capable of absorbing many times their own weight in polar liquids. These are primarily water or aqueous solutions. When absorbing the liquid, the superabsorbent swells and forms a hydrogel. Hydrogels can be formed from any cross-linked polar polymer (e.g., polyacrylamide, polyvinylpyrrolidone, amylopectin, gelatin, cellulose). However, a copolymer of acrylic acid (propenoic acid, H2C=CH-COOH) or sodium acrylate (sodium salt of acrylic acid, H2C=CH-COONa) and acrylamide is most commonly used, although the ratio of the two monomers can vary. Additionally, a core cross-linker (CXL) is added to the monomer solution, which connects the formed long-chain polymer molecules to one another through chemical bridges, also known as cross-linking. These bridges make the polymer insoluble in water.This so-called base polymer may be subjected to a process called surface cross-linking (SXL). This involves applying another chemical to the surface of the particles, which, when heated, creates a second network only on the outer layer of the grain. This shell supports the swollen gel, ensuring it stays together even under external stress (movement, pressure).

[0013] The product is conventionally used as white granules with particle sizes ranging from 100 to 1000 μm. It is mainly used in baby diapers, sanitary pads, incontinence care, in bandages, and in small quantities in cable sheathing for deep-sea cables. Other applications include so-called gel beds, gel-forming extinguishing agents in firefighting, as a mechanical stabilizer for cut flowers in vases, or as an additive for potting soil to permanently store water. However, in these cases, potassium hydroxide-neutralized acrylic acid is used due to its better environmental compatibility. In the form of spherical particles, the use of superabsorbents is known as toys under names such as "water beads," "aqua beads," or "water beads." These are superabsorbents that are commercially available in the form of spheres of variable size (submillimeters to centimeters).

[0014] The invention described here was based on the following unexpected observation. Commercially available so-called water beads (commercially available under the names Aqua Beads, Water Beads, Wasserbeads, or Gelperlen, among others) were added to a 1-liter volume of liquid. These water beads are made of a superabsorbent material. The liquid was surface water taken from a firefighting pond containing suspended matter. After an incubation period, the beads swelled to several times their original volume. The volume of the liquid portion of the mixture of the liquid and the water beads decreased. Surprisingly, it was found that the suspended matter in the liquid was not absorbed by the swelling beads. The liquid portion of the mixture, including the suspended matter (volume 400 ml), was transferred to a new container.A sample with a volume of 50 ml was taken from this liquid portion.

[0015] A 50 ml reference sample was taken directly from the liquid, i.e., the aforementioned surface water, without prior concentration according to the described procedure. Both samples were centrifuged at 5000 xg for 10 min. The supernatant was removed, and the sediment was used for nucleic acid extraction. Nucleic acid extraction was performed using a commercial kit (innuprep Stool DNA Mini Kit; IST Innuscreen GmbH). The DNA from both samples was then analyzed for total bacterial count using real-time PCR.

[0016] This showed that fewer bacteria were detected in the untreated control sample than in the sample concentrated with the superabsorbent. This means that the bacteria contained in the liquid were not absorbed into the water beads. They were concentrated and became part of the remaining liquid portion of the mixture of the liquid and the superabsorbent beads. These observations could subsequently be confirmed for other biomolecules (viruses or eukaryotic cells): After adding the superabsorbent to a volume of liquid containing these biomolecules, the volume was concentrated, and the biomolecules were concentrated in the remaining liquid. Even more surprising was that the described method can also be used to concentrate proteins and free genomic DNA, which are present in low concentrations in an aqueous solution.

[0017] On the basis of this observation, the problem underlying the invention could be solved.

[0018] The method according to the invention for the automated online detection of at least one biological target substance in a sample liquid by means of an online analysis device comprises:

[0019] - Producing concentrated sample liquid from at least a first initial volume of the sample liquid by: a. Dosing and transporting a first initial volume of the sample liquid via a liquid supply line into a first concentrator chamber of a concentrator unit; and b. Reducing the first initial volume of the sample liquid in the concentrator unit and thereby concentrating the at least one biological target substance contained in the first volume of the sample liquid by means of a first amount of a superabsorbent, wherein the reduction and the thereby concentrating are carried out by incubating a mixture formed from the first initial volume of the sample liquid with the first amount of the superabsorbent (24);

[0020] Transporting at least a portion of the generated concentrated sample liquid from the concentrator unit (3) into a detection unit of the online analysis device.

[0021] The sample liquid obtained after incubation with the superabsorbent, reduced in volume and with a correspondingly increased concentration of the biomolecules or at least one biological target substance contained therein, is also referred to here and below as “concentrated sample liquid”.

[0022] By reducing the initial volume of the sample liquid after incubation with the superabsorbent material to an arbitrarily adjustable volume, which is accompanied by an increase in the concentration of the biological target substance in the remaining volume of the sample liquid, a comparatively high proportion of the biomolecules obtained from the first initial volume of the sample liquid can be concentrated in a small volume of liquid compared to the method described in the prior art, in which biomolecules are released after binding to a filter medium by rinsing the filter medium. Thus, the method according to the invention not only brings about an efficient concentration of the target substance to be determined in the sample liquid, but also makes it possible to determine the desired substance for subsequent analysis, e.g.The volume to be extracted from a process can be selected to be comparatively small. This makes the method universally suitable for monitoring liquids in a wide variety of industrial processes, regardless of whether the volume of sample liquid transported into the process unit must be discarded and possibly disposed of after enrichment, or whether it can be returned to the process.

[0023] The target substance can, for example, be a biomolecule. The biomolecule can be selected from the group consisting of: eukaryotic cells, components of eukaryotic cells, prokaryotic cells, components of prokaryotic cells, subcellular vesicles, bacteriophages, viruses or virus components, toxins, antibodies, nucleic acids, and proteins. Any aqueous solution can be considered as a sample fluid, e.g., water and wastewater samples, fermentation fluids, fluids from food, chemical, pharmaceutical, or biochemical production processes.

[0024] The superabsorbent can comprise a plastic that absorbs water molecules to form a hydrogel. The superabsorbent can, for example, be one of the materials mentioned above. Advantageously, the plastic absorbs water or other polar solvent molecules, but not biomolecules, in particular not the target substance. This is the case, for example, with the materials mentioned above and with the superabsorbent spheres commercially available under the names “water pearls”, “aqua beads” or “water beads”. The superabsorbent can, for example, be used in the form of particles, e.g. as a powder, as granules or in the form of geometric bodies, in particular spheres. A suitable diameter of the particles, in particular of the spheres, is 100 to 5000 pm.

[0025] According to the invention, a qualitative or quantitative determination of the biological target substance present in the concentrated sample liquid is then carried out in the detection unit by means of a sensor, by means of an immunological detection method, by means of a measurement of an amount of adenosine triphosphate (ATP) present in the sample liquid, by means of a flow cytometric method, by means of a nucleic acid hybridization method and / or by means of a detection of metabolic and / or enzymatic activity of microorganisms.

[0026] The sample fluid concentrated with superabsorbents using the method according to the invention can thus be used in a variety of detection and analysis methods. It will be clear to those skilled in the art that the concentrated sample fluid can be analyzed using a variety of other detection and analysis methods not listed here.

[0027] According to an advantageous embodiment of the method according to the invention, it is provided that in the case of a flow cytometric method, the biological target substance present in the concentrated sample liquid is detected by means of an autofluorescence measurement or by means of a fluorescent label.

[0028] According to an advantageous embodiment of the method according to the invention, it is provided that in the case of a flow cytometric method, the biological target substance present in the concentrated sample liquid is detected by means of a combination of fluorescence measurement and particle scattering.

[0029] Both designs allow a statement to be made about the amount of biological target substance present in the sample. When using a sensor, the sensor can be designed based on a variety of physical and / or chemical measurement principles, including:

[0030] A sensor based on time-resolved fluorescence measurement, where background fluorescence is removed by a time gate; a sensor based on Raman spectroscopy; an electrochemical sensor, a sensor with an activated surface that allows the binding of biomolecules, where the binding can be detected optically or electrically; a color change sensor; a sensor based on absorption spectroscopy.

[0031] In case the sensor is based on absorption spectroscopy, for example, an attenuated total reflection (ATR) method, a photothermal method or a transmission spectroscopy method can be used.

[0032] According to an advantageous embodiment of the method according to the invention, labeled probes are used when the qualitative or quantitative determination is carried out using a nucleic acid hybridization method. Common methods, such as in situ hybridization for the detection of RNA or DNA sequences, can be used.

[0033] According to an advantageous embodiment of the method according to the invention, it is provided that the detection of the biomolecules, preferably bacteria, of the concentrated sample present in the detection unit is carried out by means of a measurement of metabolic or enzymatic activity of microorganisms.

[0034] For example, if a sample is to be tested for coliform bacteria, it is intended that the measurement of enzymatic activity be carried out by measuring beta-D-glucuronidase activity. Coliform bacteria, or bacteria, fecal coliforms, or thermotolerant coliforms, are lactose-splitting, gram-negative, facultatively anaerobic, rod-shaped bacteria that produce acid and gases within 48 hours at 35°C. These include the genera Citrobacter, Enterobacter, Escherichia, and Klebsiella. They are indicator organisms for the sanitary quality of water and a hygiene indicator in food production. Beta-D-glucuronidase (or ß-glucuronidase, abbreviated GUS) is a bacterial enzyme that catalyzes the hydrolysis of glucuronic acid glycosides (glucuronides). Based on the cleavage products (orFrom the resulting amount, a statement can be made about the enzymatic activity and, from this, the presence and even the quantity of microorganisms. The generation of concentrated sample liquid can involve dosing the first initial volume of sample liquid to the first amount of superabsorbent present in the first concentrator chamber or dosing the first amount of superabsorbent to the first initial volume of sample liquid present in the first concentrator chamber.

[0035] The incubation of the mixture formed from the first initial volume of sample liquid with the first amount of superabsorbent can be carried out over a predetermined, in particular selectable, period of time. In general, the achievable increase in concentration of a target substance in the sample liquid within a given period of time depends on the type of superabsorbent, the particle size of the superabsorbent, the size of the first amount of superabsorbent added, the temperature prevailing during incubation, and the length of the selected incubation period. The period of incubation can be monitored using control electronics. Alternatively, it is also possible to monitor the volume of the liquid portion and to terminate incubation when a desired, predetermined target volume is reached.

[0036] The concentrator chamber and the mixture contained therein can be temperature-controlled during incubation using a temperature control device of the concentrator unit. The temperature control device can be a heating and / or cooling device that can be operated automatically using control electronics to control and / or regulate the temperature of the mixture present in the concentrator chamber.

[0037] The method can further comprise the following step: after incubation, removing at least a portion of the concentrated sample liquid from the first concentrator chamber. The removed liquid can be fed directly to a detection unit of an analytical device. Alternatively, as described below, the concentrated sample liquid can first be fed to a collection vessel. This allows the target substance to be concentrated in several initial liquid volumes, e.g. taken as samples from a process or a body of water, using the described method and then combined for further detection. The combined concentrated samples can optionally be further concentrated again using the described method. This procedure is particularly advantageous when the concentration of the target substance in the original, untreated sample liquid is particularly low.The procedure is described in more detail below.

[0038] The sample liquid extracted from the concentrator chamber in a first stage can be transported to a collection vessel. Subsequently, any remaining concentrated sample liquid and the initial amount of superabsorbent can be removed from the concentrator chamber, e.g., by flushing the concentrator chamber with a flushing fluid, such as a flushing liquid or a flushing gas.

[0039] The procedure may further include the following steps:

[0040] - Dosing and transporting a second initial volume of the sample liquid via a liquid supply line into the first concentrator chamber of the concentrator unit; and

[0041] - producing concentrated sample liquid by reducing the second initial volume of the sample liquid in the concentrator unit and concentrating biomolecules contained in the second initial volume of the sample liquid by means of a second amount of the superabsorbent, wherein the reduction and the concentrating are carried out by incubating a mixture formed from the second initial volume of the sample liquid with the second amount of the superabsorbent, and

[0042] - Transporting at least a portion of the concentrated sample liquid obtained by reducing the second initial volume of sample liquid from the first concentrator chamber into the collection vessel.

[0043] In this way, multiple batches of concentrated sample fluid can be generated and combined in the collection vessel. This allows for the concentration of a very large volume of sample.

[0044] At least a portion of the concentrated sample liquid from the collection vessel can be metered and transported into the first concentrator chamber or into a second concentrator chamber separate from the first concentrator chamber for further concentration of the target substance using a third amount of superabsorbent. This further concentration occurs in a manner entirely analogous to that described for the first concentration, namely by creating a mixture of the concentrated sample liquid and the third amount of superabsorbent and incubating the mixture, whereby the volume of the concentrated sample liquid is again reduced and the contained biomolecules, including the target substance, are further concentrated. The further concentrated sample liquid thus obtained can be reconcentrated in the same way or fed directly to a detection unit of an analytical device for further analysis.In this way, the sensitivity of the procedure can be further increased.

[0045] The method, in all its variants described above, can advantageously be carried out fully automated by control electronics. For this purpose, the concentrator unit can have means for transporting liquids and the superabsorbent within the concentrator unit, which can include, for example, pumps and / or valves. Qualitative or quantitative determination can also be performed by amplification. For this purpose, the following is provided:

[0046] - releasing and / or isolating nucleic acids from the biomolecules contained in the concentrated sample liquid, in particular the at least one biological target substance, and producing a solution comprising the released and / or isolated nucleic acids,

[0047] - Amplifying at least one target nucleic acid contained in the solution, and

[0048] - Acquiring a measurement signal that represents a measured variable dependent on the progress of amplification and / or on the number of copies of the target nucleic acid. The control electronics of the analyzer use the acquired measurement signal to perform the qualitative or quantitative determination of the target substance in the concentrated sample liquid and / or in the sample liquid before concentration. The target nucleic acid to be amplified can be either the target substance to be detected or a nucleic acid of the target substance to be detected.

[0049] The release and / or isolation of nucleic acids from the biomolecules contained in the concentrated sample fluid and the generation of the solution comprising the released and / or isolated nucleic acids are carried out in a first microfluidic unit of the detection unit. The solution is transported to a second microfluidic unit that is fluidically connectable to the first microfluidic unit, and the amplification and acquisition of the measurement signal are carried out in the second microfluidic unit. The first and second microfluidic units can be housed in two separate cartridges or in a single cartridge.

[0050] The release and / or isolation of nucleic acids from the biomolecules contained in the concentrated sample liquid and the generation of the solution comprising the released and / or isolated nucleic acids may comprise at least the following steps: - Adding one or more lysis reagents to the liquid sample in order to release nucleic acids of the biomolecules,

[0051] - binding the released nucleic acids to a nucleic acid binding material; and - washing the bound nucleic acids with a washing solution, and

[0052] - Detaching the nucleic acids from the nucleic acid-binding material by elution. The resulting eluate forms the solution containing the released and / or isolated nucleic acids.

[0053] The release and / or isolation of nucleic acids from the biomolecules contained in the concentrated sample liquid and the generation of the solution comprising the released and / or isolated nucleic acids may comprise the following steps:

[0054] - thermal treatment of the biomolecules contained in the concentrated sample fluid in a buffer solution, for example, in a phosphate-buffered saline solution, in water, in a Tris buffer, or in a buffer containing a low concentration of a detergent. In this case, the buffer solution forms the aforementioned solution containing the released and / or isolated nucleic acids with the nucleic acids released by the thermal treatment.

[0055] Thermal release can involve dispersing the sample in the buffered solution and incubating it. This process variant is suitable, for example, for applications in which the target substance to be determined is a nucleic acid that is already present freely in the liquid to be analyzed. Thus, conventional lysis can be omitted. The process can also be used if the target substance is a bacterium or a virus. In this case, the biomolecules contained in the sample can be thermally / chemically destroyed, and the nucleic acid contained can be released.

[0056] The amplification, for example, carried out in the second microfluidic unit, can be carried out using conventional PCR-based methods. The qualitative or quantitative determination of the target substance can also be carried out in a conventional manner by using a sensor to record at least one measured value of a measurand at a specific time or at several time points, which correlates with the number of copies of a target nucleic acid generated by the amplification at that time. From the obtained measured value or measured value curve as a function of time, the presence of the target nucleic acid and thus of the biological target substance in the sample fluid can be qualitatively detected or a quantitative value, e.g.a concentration of the biological target substance in the original sample liquid or in the sample liquid after concentration using the method described above is determined and output by the control electronics as a measurement result.

[0057] All steps of the method according to the invention described here for the online detection of at least one biological target substance in a sample fluid, in all specified embodiments, can be carried out automatically using the control electronics of the online analysis device. For this purpose, it can, for example, effect liquid transport (sample fluid and / or reagents) or solid transport (superabsorbent material) by actuating controllable valves and / or pumps, and control and / or regulate the incubation time and temperature during concentration and / or release of nucleic acids using a predefined sequence program.

[0058] The invention also comprises a concentrator unit for the automated concentration of a target substance in a sample liquid, in particular after the first method step of the method described above, comprising:

[0059] - a first concentrator chamber; - at least one liquid supply line opening into the first concentrator chamber; and

[0060] - at least one liquid discharge line opening into the first concentrator chamber, wherein the concentrator unit is configured to add a first amount of a superabsorbent to an initial liquid volume of the sample liquid introduced into the first concentrator chamber via the liquid supply line, or is configured to add the initial liquid volume to the first amount of the superabsorbent.

[0061] The initial amount of superabsorbent can be placed in the first concentrator chamber. The concentrator chamber can be designed as a replaceable cartridge. This allows the replaceable cartridge containing the superabsorbent to be replaced with a new one for the concentration of the target substance in each new batch of sample liquid.

[0062] Alternatively, the concentrator unit can have at least one reservoir or be connected to a reservoir containing superabsorbent, particularly in the form of particles. Preferably, one of the above-mentioned materials, particularly in spherical form, is used as the superabsorbent. Commercially available superabsorbent spheres with a diameter of approximately 1 mm, which are also commercially available under the name "water beads," "aqua beads," "water beads," or "gel beads," have proven to be very advantageous. Superabsorbent spheres of this size can be very well dispersed in the concentrator chamber.

[0063] The reservoir can be connected to the first concentrator chamber, for example, via a superabsorbent supply line that can be closed by a valve, in particular one that can be controlled by means of control electronics. When the valve is open, at least a portion of the superabsorbent contained in the reservoir enters the first concentrator chamber as the first amount of superabsorbent. The transport of superabsorbent can be effected by gravity. However, it is also possible for the concentrator unit to have means for transporting the superabsorbent, which may be present, for example, in particle form, e.g., a pump and / or means for generating a gas stream that transports the superabsorbent particles.For dosing and transporting the superabsorbent, control electronics designed for automatic operation of the concentrator unit can be used. These electronics are configured to actuate the aforementioned means and / or valve to measure the first amount of superabsorbent and transport it into the concentrator chamber. These control electronics can be configured, in conjunction with pumps and / or valves, to dose the initial volume of sample liquid into the concentrator chamber.

[0064] The concentrator device may comprise means for moving, in particular stirring, the liquid contained in the reaction chamber. The means may comprise, for example, a stirrer, a supply line for an inert gas into the reaction chamber, and an outlet line for the inert gas, which are arranged such that the inert gas flows through the liquid contained in the reaction chamber, or a drive for moving the reaction chamber.

[0065] The concentrator unit may further comprise a temperature control device configured to control the temperature of the first concentrator chamber. The temperature control device may, for example, comprise heating and / or cooling elements operable by the control electronics to control and / or regulate the temperature of a liquid contained in the concentrator chamber.

[0066] The liquid drain for draining at least part of the liquid volume of the sample liquid present in the first concentrator chamber can be fluidly connectable to a collecting vessel.

[0067] The collection vessel can have a collection vessel outlet that is fluidically connectable to the first concentrator chamber. In this way, sample liquid present in the collection vessel, which has already been concentrated in a first stage, can be redirected to the first concentrator chamber for further concentration in a second stage by again adding superabsorbent to the concentrated sample liquid in the first concentrator chamber and incubating the mixture.

[0068] Alternatively, the concentrator unit may comprise an additional second concentrator chamber, wherein the liquid discharge line of the first concentrator chamber connects it, in particular via a collecting vessel, to the second concentrator chamber; and a further liquid discharge line opening into the second concentrator chamber, wherein the concentrator unit is configured to add a second amount of the superabsorbent to a volume of the sample liquid concentrated in the first concentrator chamber introduced into the second concentrator chamber or to add the liquid volume to the second amount of the superabsorbent.

[0069] The invention also encompasses an online analysis device for detecting at least one biological target substance in a sample liquid, in particular according to the method described above. The analysis device comprises: control electronics; a concentrator unit according to one of the above-described embodiments, wherein the concentrator unit has a liquid outlet for concentrated sample liquid produced in the concentrator unit from the sample liquid; and a detection unit fluidically connectable to the liquid outlet of the concentrator unit; and at least one transport device controllable by the control electronics, e.g.a pump configured to transport concentrated sample liquid from the liquid outlet of the concentrator unit into the detection unit, wherein the control electronics are configured to determine the target substance in the concentrated sample liquid and / or in the sample liquid before concentration qualitatively or quantitatively based on a molecular genetic method by means of the detection unit.

[0070] According to an advantageous embodiment of the online analysis device according to the invention, it is provided that for carrying out an immunological detection, the detection unit is designed in such a way that it allows the supply of reagents required for immunological detection via separate inlet openings

[0071] According to an advantageous embodiment of the online analysis device according to the invention, the detection unit for measuring the amount of adenosine triphosphate present in the sample fluid is designed to allow the addition of reagents required for the measurement via separate inlet openings. The measurement of intracellular ATP reliably indicates the status of the energy supply and is an important laboratory marker for detecting possible mitochondrial dysfunction. ATP can be rapidly detected, for example, by light emission through the combined use of luciferase and a luminometer.

[0072] According to an advantageous embodiment of the online analysis device according to the invention, if a nucleic acid hybridization method is carried out using labeled probes, the detection unit has separate inlet openings that allow the introduction of reagents required for the nucleic acid hybridization method. Common reagents for this purpose are primers, nucleotides, buffers, etc.

[0073] According to an advantageous embodiment of the online analysis device according to the invention, it is provided that, in the case where the measurement of enzymatic activity is carried out by means of a measurement of a beta-d-glucuronidase activity of coliform germs, the detection unit has separate inlet openings which allow the supply of reagents which are required for a measurement of the beta-d-glucuronidase activity.

[0074] The online analysis device can further comprise a sample pump, with the control electronics configured to control the sample pump to transport a predetermined volume of sample liquid into the concentrator unit. The sample supply line can be fluidically connected to a sample reservoir or directly to a body of water or a pipe in a process plant, a fermenter, or another process vessel.

[0075] The detection unit can comprise the following components: a first microfluidic unit fluidically connectable to a liquid outlet for concentrated sample liquid of the concentrator unit, which is configured to release and / or isolate nucleic acids from biomolecules contained in the concentrated sample liquid, in particular the at least one biological target substance; a second microfluidic unit connected to the first microfluidic unit, which is configured to receive an eluate comprising the released and / or isolated nucleic acids and to amplify a target nucleic acid; and a sensor, in particular an optical one, which is configured to generate a measurement signal dependent on the progress of an amplification performed in the second microfluidic unit and / or on a number of copies of the target nucleic acid in the second microfluidic unit and to output it to the control electronics;and wherein the control electronics are configured to qualitatively and / or quantitatively determine the biological target substance in the liquid based on the measurement signal output by the sensor;

[0076] The liquid outlet of the concentrator unit can be fluidically connected to the aforementioned first concentrator chamber and / or to a second concentrator chamber (if present) and / or to a likewise optionally present collection vessel. In various configurations or operating alternatives of the online analyzer, concentrated sample liquid can thus be transported directly from the first or the optionally present second concentrator chamber or via the optionally present collection vessel into the detection unit.

[0077] The first and second microfluidic units can be arranged together in a cartridge, in particular an exchangeable one. Alternatively, the first microfluidic unit can be arranged in a first cartridge and the second microfluidic unit can be arranged in a second cartridge, different from the first cartridge, wherein the first and second cartridges are fluidically connectable to one another in order to transport liquid from the first cartridge to the second. The first and second cartridges can be designed to be exchangeable.

[0078] The first microfluidic unit can be configured to receive the concentrated sample with the biomolecules it contains and to add one or more lysis reagents to release nucleic acid from the biomolecules contained in the sample. Subsequently, the released nucleic acids can be bound, optionally with the addition of a component that enhances / mediates the binding of the nucleic acid to a nucleic acid-binding material, and the nucleic acids bound to this material can be washed one or more times with a washing solution. The control electronics can be configured to control the transport of the particles and, optionally, the reagents through the first microfluidic unit.The first microfluidic unit can be further configured to separate the nucleic acids from the nucleic acid-binding material by eluting and to transport the eluate into the second microfluidic unit via a fluid line connecting the first microfluidic unit to the second microfluidic unit. The control electronics can be configured to control the eluting and transport of the eluate.

[0079] Alternatively, the first microfluidic unit can be configured to receive the concentrated sample with the contained biomolecules and to thermally / chemically release the biomolecules in a buffer solution, for example, in a phosphate-buffered saline solution (PBS buffer), water, or a Tris buffer, or optionally a buffer containing a detergent. The control electronics are configured to control the thermal release of the biomolecules and transport the buffer solution with the biomolecules or nucleic acids dissolved therein to the second microfluidic unit for subsequent amplification. This is suitable, for example, for use in an application in which the biological target substance to be detected is a nucleic acid freely present in the liquid.

[0080] The first microfluidic unit can contain the reaction components required for the release and / or isolation of the nucleic acid in solid form, for example in the form of pellets obtained by lyophilization, or in reagent chambers sealed by microvalves or in reagent packs sealed by foils, which can be opened automatically by the application of force or heat, in particular by means of the control electronics.

[0081] Reagents for amplification can be stored in the second microfluidic unit in a corresponding manner. It can also contain one or more detection chambers in which the amplification of a target nucleic acid or of several different target nucleic acids can be carried out in parallel. The second microfluidic unit can include a temperature control device for the detection chambers. The temperature control device can, for example, comprise thermoelectric elements controllable by the control electronics.

[0082] The control electronics described here and above in connection with the method according to the invention and the concentrator unit according to the invention can comprise an electronic data processing device with at least one processor and a memory in which one or more operating programs are stored, which can be executed by the processor in order to automatically control the analysis device and to determine qualitative or quantitative analysis results from the measurement signals of the sensor. The operating programs can be designed such that the control electronics controls the analysis device to carry out the method described above. The control electronics can also comprise a user interface, e.g. a touch screen or another display in combination with an input keyboard. The control electronics can be configured to communicate wirelessly by radio or via a data line with another data processing device, e.g.to be connected to a computer, a process control, a display or operating device, in particular a portable one, for communication purposes.

[0083] The automatic control of the concentration performed in the concentrator unit can be carried out using the same control electronics that also control the other components of the analyzer. Alternatively, the concentrator unit can also have its own control electronics designed to automate the concentration of the sample liquid in the manner described. The control electronics of the concentrator unit are advantageously connected for communication with the control electronics of the analyzer, which controls the other components of the analyzer, namely, in particular, the detection unit.

[0084] The analyzer can thus perform online determinations of biological target substances in a liquid without requiring manual sampling or preparation, or the transport of samples to a laboratory. In particular, the analyzer can execute sampling and detection cycles fully automatically, which can be executed, for example, event-driven by the control electronics or at predefined time intervals.

[0085] The invention is described below with reference to the exemplary embodiments illustrated in the figures. Like reference numerals designate like components of the components shown in the figures. They show:

[0086] Fig. 1 is a schematic representation of an online analysis device for the qualitative or quantitative determination of a biological target substance in a liquid;

[0087] Fig. 2 shows a first embodiment of a concentrator unit of the online analysis device shown in Fig. 1 according to a first embodiment; and

[0088] Fig. 3 shows a second embodiment of a concentrator unit for an online analysis device.

[0089] Fig. 1 schematically shows an embodiment of an online analysis device 1 for detecting a biological target substance in a sample liquid. In the present example, the analysis device 1 has a housing 2 in which all components of the device are housed. These components include a concentrator unit 3, a detection unit 4, and control electronics 5. The concentrator unit 3 enables the detection of biomolecules contained in a sample liquid even at very low concentrations, as it is designed to concentrate biological target molecules in a volume of sample liquid to be analyzed and to pass the concentrated sample liquid on to the detection unit 4 for detection and, if necessary, preparatory steps such as extraction and / or isolation of nucleic acids.

[0090] The concentrator unit 3 is fluidically connected via a sample feed line 6 to a sample reservoir 7 or to a process container, e.g., a pipeline, a reactor, or a fermenter, in which the liquid to be analyzed is contained. A pump 9 is used to transport and meter the liquid from the sample reservoir 7 into a first concentrator chamber 8 contained in the concentrator unit 3. The pump 9 can be controlled by the control electronics 5 according to an operating program stored in and executed by the control electronics 5. The first concentrator chamber 8 can also be fluidically connected to reservoirs 10, 11. The reservoirs 10, 11 contain substances that serve to enrich the biomolecules contained in the liquid. In the present example, each reservoir 10, 11 is fluidically connected to the concentrator chamber in the concentrator unit 3 via a fluid line.A pump 12, 13 is used to transport the substances contained in the reservoirs through the fluid lines. It is also possible for the reservoirs 10, 11 to be integrated directly into the concentrator unit 3. The transport of the substances can, of course, be accomplished by other means known to those skilled in the art, e.g., pneumatically or by utilizing gravity or, for example, hydrostatic pressure.

[0091] The concentrator unit 3 can optionally have a temperature control unit (not shown in Fig. 1) that serves to set a specific temperature in the concentrator chamber 8. The temperature control unit can comprise a resistance heater, a cooling unit, and / or thermoelectric elements for selective heating or cooling. It can be connected to the control electronics 5, wherein the control electronics 5 are configured to control or regulate the temperature in the concentrator chamber 8.

[0092] The concentrator unit 3 can also have a device 14 for moving or stirring a liquid or liquid mixture contained in the reaction chamber 8. In the present example, the device 14 comprises a drive for moving, e.g., shaking, the reaction chamber 8. The device 14, in particular the drive, can be controlled by the control electronics 5.

[0093] The concentrator chamber 8 can be fluidically connected via the fluid line 15 either to the detection unit 4 or to a liquid outlet 17 by means of a valve 16 actuated by the control unit 5.

[0094] In the present example, the detection unit 4 comprises an analysis cartridge in which two microfluidic units, namely a first microfluidic unit 19 and a second microfluidic unit 20, are integrated and fluidically connected to each other via the fluid line 18. In another embodiment, the microfluidic units can also be housed in separate cartridges. The analysis cartridge is replaceable in the present example.

[0095] The first microfluidic unit 19 is configured to receive a sample containing the concentrated biomolecules from the reaction chamber and to prepare it for subsequent amplification and detection in the second microfluidic unit 20. This can be done using known extraction and isolation methods. For this purpose, the first microfluidic unit 19 comprises reagents and optionally a temperature control unit for incubating reaction mixtures prepared in the microfluidic unit 19. To control the transport of the sample through the first microfluidic unit 19 and to carry out the extraction and / or isolation of nucleic acids, the detection unit 4 comprises suitable means known to those skilled in the art, which are controllable by the control electronics 5. The second microfluidic unit 20 comprises means for amplification, i.e., reagents and temperature control elements, e.g.thermoelectric elements, as well as means for transporting and aliquoting fluids within the second microfluidic unit 20. To detect the progress of the amplification in a manner known per se, e.g., using a real-time PCR-based method, the detection unit has a sensor 21, which can, for example, perform fluorescence measurements in the second microfluidic unit 20 and output measurement signals to the control electronics 5. The control electronics 5 are configured to determine a qualitative or quantitative analysis result based on the measurement signals.

[0096] The control electronics 5 can be a central processing unit, e.g., a CPU with a processor and memory and operating programs stored therein. It can also be divided into several processing units within the analyzer. For example, the concentrator unit 3 and the detection unit 4 can each have their own local electronics, which are connected to a higher-level electronics unit for communication. The higher-level electronics and the local electronics together form the control electronics 5.

[0097] Fig. 2 schematically shows a first embodiment of a concentrator unit 3 of the online analysis device 1 shown in Fig. 1 in detail. The structure of the concentrator unit 3 shown in Fig. 2 is merely one possible embodiment. Those skilled in the art can devise numerous variants without deviating from the inventive concept.

[0098] The concentrator unit 3 has a concentrator chamber 8 with various inlets and outlets. The supply line 23 is connected to a storage container serving as a reservoir 10, which contains a superabsorbent 24, e.g., in the form of spheres made of the superabsorbent material. The superabsorbent 24 is added to the concentrator chamber 8 via the supply line 23. A first opening 25 serves to supply the sample liquid. A specific initial volume of the sample liquid is introduced into the concentrator chamber 8 via this opening. A second opening 26 is connected to the fluid line connecting the reaction chamber (1) to a sample outlet and the detection unit.The second opening 26 can have a diameter that is smaller than the diameter of the superabsorbent spheres, so that only the liquid portion of the mixture of sample liquid and adsorbent can be discharged from the concentrator chamber 8 via the second opening 26 toward the detection unit 4. Optionally, the second opening 26 can also have a size selector, e.g., a grid or a filter, that retains the superabsorbent spheres in the concentrator chamber 8.

[0099] The superabsorbent contained in the concentrator chamber 8 and residues of the sample liquid can be removed from the concentrator chamber 8 through a third opening 27 serving as a drain, which in the present example is opposite the opening 25 through which the sample liquid can be introduced into the concentrator chamber 8. To completely remove the superabsorbent 24 and the residues of the sample liquid, a rinsing liquid or a rinsing gas can be passed through the concentrator chamber 8. In the present example, the rinsing liquid can either be the sample liquid that is passed from the sample reservoir 7 through the concentrator chamber 8. Alternatively, the rinsing liquid can be a liquid stored in the reservoir 11. In the latter case, the reservoir 11 is fluidically connected to the first opening 25. Optionally, valves that can be actuated by the control electronics 5 are arranged in the respective supply lines.

[0100] The control electronics 5 may comprise an operating program which it can execute to carry out the following procedure:

[0101] 1) Introducing a first initial sample liquid volume from the sample reservoir 7 into the concentrator chamber 8,

[0102] 2) Adding a first amount of the superabsorbent 24 from the reservoir 10 into the concentrator chamber 8 to the first initial sample liquid volume,

[0103] 3) Incubating the mixture of the superabsorbent 24 and the sample liquid for a period of time measured by the control electronics 5, whereby the volume of the liquid portion of the mixture is reduced while concentrating biomolecules, in particular a biological target substance to be determined in the liquid portion,

[0104] 4) After the incubation period has elapsed, drain the liquid portion.

[0105] As already described above, the superabsorbent absorbs polar solvents, in the present embodiment, for example water, from the sample liquid to form a gel. Biomolecules, in particular the biological target substance to be determined, are not absorbed by the superabsorbent. During incubation, the volume of the liquid portion of the mixture of superabsorbent and sample liquid decreases, and the target substance is concentrated in the liquid. The incubation time can be selected to ensure that the concentration of the target substance in the concentrated sample liquid after incubation lies above a threshold value, e.g. above a detection limit predetermined by the detection unit. The time until the desired volume reduction orThe desired concentration depends on the following factors: type of superabsorbent, particle size of the superabsorbent particles, amount of superabsorbent added, and temperature during incubation. The appropriate amount of superabsorbent and incubation time for a specific type of sample liquid can be determined through preliminary tests and stored in the control electronics.

[0106] The control unit 5 can be configured to adjust or regulate the temperature of the liquid contained in the concentrator chamber 8 or of the mixture of sample liquid and superabsorbent to a constant value using the aforementioned temperature control device. All of these steps can be performed automatically using the control unit 5. The liquid portion is a concentrated sample liquid, i.e., sample liquid that contains the biological target substance in an increased concentration due to the volume reduction caused by the absorption of water by the superabsorbent. In one possible embodiment, the concentrated sample liquid can be transported directly into the detection unit 4 according to the exemplary embodiment described here.Using the method described here and the concentrator unit 3 used to carry out this method, it is possible to reduce a large-volume water sample to a much smaller sample volume and, correspondingly, achieve a significant increase in the concentration of biomolecules. This facilitates and / or accelerates subsequent detection using molecular genetic methods. If the concentration of biomolecules in the sample liquid is initially very low, the method described here can even enable molecular genetic detection of the target substance in the first place.

[0107] A further advantage arises from the fact that the inventive approach makes it possible to concentrate bacteria, viruses, and free nucleic acids completely non-selectively. Accordingly, it is also very simple to use the method to first concentrate, extract, and subsequently qualitatively or quantitatively determine several different biological target substances. For example, the qualitative or quantitative determination of the biological target substance present in the concentrated sample fluid is carried out in the detection unit 4 by means of a sensor, by means of an immunological detection method, by measuring the amount of adenosine triphosphate present in the sample fluid, by means of a flow cytometric method, by means of a nucleic acid hybridization method, and / or by means of detecting the metabolic and / or enzymatic activity of microorganisms.

[0108] The opening 26 of the concentrator unit 3 is fluidically connected to the fluid line 15 of the analysis device 1, which can be connected via the valve 16 to the detection unit 4 on the one hand and to the liquid outlet 17 on the other. The target substance can also be detected by PCR, as described in more detail below: The concentrated sample liquid is transported via the fluid line 15 into the first microfluidic unit 19 by means of the control electronics 5. A further pump controlled by the control electronics 5 can be used for this purpose. The valve 16 is also actuated by the control electronics 5. Automated nucleic acid extraction and / or isolation takes place in the first microfluidic unit 19, while automated amplification and detection by PCR or real-time PCR takes place in the second microfluidic unit 20, which is connected downstream of the first microfluidic unit 19.Microfluidic units suitable for automated nucleic acid extraction, amplification, and detection for the method described here are generally known in the prior art. A centrifugal platform, such as that described in WO 2013 / 045631 A1 and EP 2 621 632 A1, is advantageous for the present application. However, the use of other microfluidic platforms or lab-on-chip systems that provide liquid and reagent transport using capillary forces, pumps, or pneumatics instead of centrifugal force is also possible.

[0109] The extraction and / or isolation of the nucleic acids in the first microfluidic unit 19 is carried out using known reagents and methods, as already described in the introduction. If the biological target substance is not nucleic acid already present in the liquid taken from the sample, the biomolecules contained in the concentrated sample liquid are lysed. The free nucleic acids are bound to a nucleic acid-binding material, e.g., in particle form. The nucleic acid-binding material with the bound nucleic acids is subsequently washed in a known manner, and the nucleic acids are finally detached from the material. The eluate containing the dissolved nucleic acids is transported to the second microfluidic unit 20, again controlled by the control electronics 5.

[0110] In the second microfluidic unit 20, specific volumes of the eluate are transported to one or more detection chambers, and amplification reagents are added. Amplification then takes place in the detection chambers, the progress of which is monitored by fluorescence measurements with the sensor 21. The sensor 21 outputs measurement signals to the control electronics 5. From the measurement signals, the control electronics determines either a qualitative value that indicates whether the specific biological target substance is present in the liquid taken from the sample reservoir 7. Alternatively or additionally, the control electronics 5 can also determine a quantitative value from the measurement signals that represents a concentration of the biological target substance in the liquid.In an advantageous embodiment, different target nucleic acids can be amplified in several detection chambers, allowing several different biological target substances in the liquid to be qualitatively or quantitatively determined in parallel. Before or after the transfer of the concentrated liquid from the concentrator unit 3 to the detection unit 4, or after detection has taken place, the liquid remaining in the concentrator unit 3 is discharged from the process unit 3 through the liquid outlet 17. The concentrator unit 3 can be sufficiently rinsed with a rinsing medium, for example, with liquid aspirated from the sample reservoir 7, and the process can now be repeated for another measurement. The microfluidic units 19, 20 in the detection unit can be replaced with new microfluidic units 19, 20 for further measurements.All steps described here can be carried out automatically by the control electronics 5.

[0111] Fig. 3 schematically shows an alternative embodiment of a concentrator unit 33 which can be used in the analysis device of Fig. 1 instead of the concentrator unit 3 shown in Fig. 2. The concentrator unit 33 is designed to concentrate a sample liquid in several stages and thus allows the concentration even of target substances which are present in only an extremely low concentration in the original sample liquid. The concentrator unit 33 has a first concentrator chamber 8 which has an inlet 25 for sample liquid, a supply line 23 for superabsorbent, e.g. in the form of spheres with a diameter of the order of 1 mm, and two outlet lines 26, 27. The supply line 23 for the superabsorbent can be connected to a reservoir 10 which contains a supply of superabsorbent.The first discharge line 26 from the concentrator chamber 8 leads to a collecting vessel 28, the second discharge line 27 serves as a discharge line for used liquid and used superabsorbent and can be connected, for example, to a waste collection container.

[0112] The collection vessel 28 is fluidically connected to a second concentrator chamber 30 via a liquid drain line 29. The second concentrator chamber 30 can be configured identically to the first concentrator chamber 8. In the present example, it has an additional supply line 31 for superabsorbent, which is fluidically connected to the same reservoir 10 of the analyzer as the supply line 23 to the first concentrator chamber 8. The second concentrator chamber has a first drain line 15, which is fluidically connected to the detector unit 4 of the analyzer in order to supply concentrated sample liquid to the detector unit for the qualitative and / or quantitative detection of the biological target substance. The concentrator chamber 8 has a second drain line 32 for draining used liquid and / or used superabsorbent.

[0113] A multi-stage concentration of a target substance in a sample liquid can be carried out by means of the concentrator unit 33, in particular automated by means of control electronics, e.g. the control electronics 5 of the analysis device, in the following manner: In a first stage, a first initial volume of the sample liquid to be analyzed can be introduced into the first concentrator chamber 8. After adding an initial amount of the superabsorbent, the mixture thus formed can be incubated for a predetermined period of time or until a certain final volume of the liquid portion is reached. At least a portion of the liquid portion remaining after incubation can then be passed from the concentrator chamber 8 into the collecting vessel 28 via the first discharge line 26. Residual liquid and used superabsorbent can be discharged from the concentrator chamber via the discharge line 27.

[0114] In the same way, one or more additional initial volumes of the sample liquid can be reduced in the first concentrator chamber 8 in order to concentrate the target substance in these additional volumes. After the respective incubation, at least a portion of the remaining liquid portion is transferred to the collection vessel 28.

[0115] The collected sample liquid, already concentrated in a first stage, can be further concentrated in a second stage using the second concentrator chamber 30. For this purpose, at least a portion of the concentrated sample liquid contained in the collection vessel 28 can be introduced into the second concentrator chamber 30 via the discharge line 29 of the collection vessel 28. A second amount of superabsorbent is introduced into the second concentrator chamber 30 via the superabsorbent supply line 31. The mixture thus formed is incubated in the second concentrator chamber 30 for a predetermined period of time and / or until a desired volume of the liquid portion is reached. At least a portion of the liquid portion can then be fed to the detector unit 4 of the analyzer via the liquid line 15. Unneeded liquid and / or the used superabsorbent can be drained from the second concentrator chamber 30 via the discharge line 32.

[0116] All steps of the method described here can be carried out fully automatically by control electronics, e.g., the control electronics 5 of the analyzer 1. For this purpose, the concentrator unit has suitable means for transporting liquid and superabsorbent particles through the fluid lines into the various chambers, in particular pumps and valves (not shown in Fig. 3), which the control electronics actuate to carry out the described method for concentrating the target substance in the sample liquid.

[0117] The analytical device and method according to the invention thus ideally solves the problem by linking available detection systems and enables the performance of online liquid analysis of biological targets, which appears particularly advantageous for the monitoring of water or wastewater in networks, sewage treatment plants, or treatment facilities, as well as bodies of water. The method according to the invention makes it possible for the first time to implement molecular genetic monitoring of biological targets in a practical and cost-effective manner without manual intervention, starting with a large-volume sample and ending with detection.

Claims

Patent claims 1. Method for automated online detection of at least one biological target substance in a sample liquid using an online analyzer (1), comprising: Producing concentrated sample liquid from at least one initial volume of sample liquid by: c. dosing and transporting a first initial volume of sample liquid via a liquid feed line (25) into a first concentrator chamber (8) of a concentrator unit (3, 33); and d. reducing the first initial volume of sample liquid in the concentrator unit (3, 33) and thereby concentrating the at least one biological target substance contained in the first volume of sample liquid by means of a first quantity of a superabsorbent (24), wherein the reduction and the associated concentration are carried out by incubating a mixture formed from the first initial volume of sample liquid with the first quantity of the superabsorbent (24); Transporting at least a portion of the generated concentrated sample liquid from the concentrator unit (3) to a detection unit (4) of the online analyzer; qualitative or quantitative determination of the biological target substance present in the concentrated sample liquid in the detection unit (4) by means of a sensor, by means of an immunological detection method, by means of measuring the amount of adenosine triphosphate present in the sample liquid, by means of a flow cytometric method, by means of a nucleic acid hybridization method and / or by means of detecting the metabolic and / or enzymatic activity of microorganisms.

2. Method according to claim 1, wherein in the case of a flow cytometric method the biological target substance present in the concentrated sample liquid is detected by means of an autofluorescence measurement or by means of a fluorescent label.

3. Method according to 1 or 2, wherein in the case of a flow cytometric method the biological target substance present in the concentrated sample liquid is detected by means of a combination of fluorescence measurement and particle scattering.

4. Method according to claim 1, wherein the sensor is based on a time-resolved fluorescence measurement and the background fluorescence is removed by a time gate.

5. The method of claim 1, wherein the sensor is based on Raman spectroscopy.

6. Method according to claim 1, wherein an electrochemical sensor is used as the sensor.

7. Method according to claim 1, wherein a sensor with an activated surface is used as the sensor, which surface allows the binding of biomolecules, wherein the binding can be detected optically or electrically.

8. Method according to claim 1, wherein a color change sensor is used as the sensor.

9. Method according to claim 1, wherein the sensor is based on absorption spectroscopy.

10. The method of claim 9, wherein an attenuated total reflection (ATR) method, a photothermal method or the transmission spectroscopy method is used for absorption spectroscopy.

11. Method according to claim 1, wherein labeled probes are used for carrying out a nucleic acid hybridization process.

12. Method according to claim 1, wherein the detection of the biomolecules, preferably bacteria, of the concentrated sample present in the detection unit (4) is carried out by measuring the metabolic or enzymatic activity of microorganisms.

13. Method according to claim 12, wherein the measurement of enzymatic activity is carried out by measuring the beta-d-glucuronidase activity of coliform bacteria.

14. Concentrator unit (3, 33) for the automated execution of a concentration of a target substance in a sample liquid according to the first process step of the process according to one of claims 1 to 13, comprising: a first concentrator chamber (8); at least one liquid feed line opening into the first concentrator chamber (8). (25); and at least one liquid outlet opening into the first concentrator chamber (8) (26); wherein the concentrator unit (3, 33) is configured to add an initial quantity of a superabsorbent (24) to an initial liquid volume of the sample liquid introduced into the first concentrator chamber (8) via the liquid inlet (25), or is configured to add the initial liquid volume to the first quantity of the superabsorbent (4).

15. Online analyzer (1) for detecting at least one biological target substance in a sample liquid according to the method of any one of claims 1 to 13, comprising: a control electronics unit (5); a concentrator unit (3, 33) according to claim 14, wherein the concentrator unit (3, 33) has a liquid outlet for concentrated sample liquid produced in the concentrator unit (3, 33) from the sample liquid; and a detection unit (4) fluidically connectable to the liquid outlet of the concentrator unit (3, 33); and at least one transport device controllable by the control electronics unit (5), e.g. a pump, which is configured to transport concentrated sample liquid from the liquid outlet of the concentrator unit (3, 33) to the detection unit (4), wherein the control electronics unit (5) is configured to determine the target substance in the concentrated sample liquid and / or in the sample liquid before concentration qualitatively or quantitatively based on a molecular genetic method by means of the detection unit (4).Online analyzer according to claim 15, wherein, for carrying out immunological detection, the detection unit (4) is designed to allow the supply of reagents required for immunological detection via separate inlet openings. Online analyzer according to claim 15, wherein, for carrying out the measurement of the amount of adenosine triphosphate present in the sample liquid, the detection unit (4) is designed to allow the supply of reagents required for the measurement via separate inlet openings. Online analyzer according to claim 15, wherein, in the case that a nucleic acid hybridization procedure is carried out using labeled probes, the detection unit (4) has separate inlet openings which allow the supply of reagents required for the nucleic acid hybridization procedure.Online analyzer according to claim 15, wherein, in the case that the measurement of enzymatic activity is carried out by means of a measurement of beta-d-glucuronidase activity of coliform bacteria, the detection unit (4) has separate inlet openings which allow the supply of reagents which are required for a measurement of the beta-d-glucuronidase activity.

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