DE102022113316A1 - Method for the automated online detection of at least one biological target substance in a sample liquid using an online analysis device - Google Patents

Abstract

The invention relates to a method for the automated online detection of at least one biological target substance in a sample liquid by means of an online analysis device (1), comprising: - generating concentrated sample liquid from at least a first initial volume of the sample liquid by means of: e. Dosing and transporting 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); andf. Reducing 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 contained in the first volume of the sample liquid by means of a first amount of a superabsorber (24), the reduction and the associated concentration by incubating a mixture formed from the first initial volume of the sample liquid with the first amount of the superabsorbent (24); - Transporting at least part of the concentrated sample liquid produced from the concentrator unit (3) into a detection unit (4) of the online analysis device ,Qualitative or quantitative determination of 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 measuring an 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 detecting the metabolic and/or enzymatic activity of microorganisms. The invention also relates to a device for carrying out the method.

Description

The invention relates to a method for the automated online detection of at least one biological target substance using 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 using molecular genetic methods in a sample liquid.

The detection of biological parameters plays an increasingly important role in the process industry, for example in the monitoring and purification of industrial water or sewage water, in food production and in the pharmaceutical and life science sector, for example in the monitoring of biotechnological processes carried out in fermentors. Target substances to be detected or monitored can be, for example, viruses, bacteria and plasmid-associated bacterial resistance genes.

The detection of such biological target substances in liquids can be done 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 that have the necessary equipment systems. For automated sampling from processes, e.g. from clarification tanks, fermenters, production vessels or lines, manually handled or automatic sampling devices and sample collectors are known. 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 is carried out manually or at least partially automatically.

For example, in the field of water and wastewater analysis, online analysis devices are known which carry out a fully automated sampling and quantitative determination of a, usually inorganic, analyte in the sample. Such an online analysis can be carried out continuously or discontinuously.

Out of US 2015/0224502 A1 is a sample collection device for flow sampling in water. The device has several flow-through sample cartridges which are designed to absorb water from the body of water to be monitored and, if necessary, to retain samples of components of the liquid or solids in a filter or adsorption medium, while the absorbed water is released from the cartridges back into the body of water is returned. 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 lysate and made available to an analysis module for further analysis, for example using qPCR.

With low concentrations of the target substances in the liquid to be analyzed, the problem arises of providing a sufficient amount of the target substance for the subsequent analysis when taking a sample. With flow sampling as in US 2015/0224502 A1 As described, dissolved substances or solids present in the water in low concentrations can be retained in the filter medium by passing a correspondingly large volume of water through. However, this is only practical if the liquid to be analyzed can be returned to the medium after the sample without any restrictions, as is the case, for example, with water analysis. When monitoring processes, for example in water treatment, the food industry or the pharmaceutical sector, it is often not possible to reintroduce the removed liquid. There is a need here 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.

Another disadvantage of the in US 2015/0224502 A1 The device described is that the substance retained in the filter or adsorption medium is passed into the analysis module by lysis and elution from the filter or adsorption medium. On the one hand, it is to be expected that only a relatively small proportion of the target substance from the filter or adsorption medium reaches the analysis module, and on the other hand, a certain minimum volume of liquid is required to rinse out the filter, so that the solution entering the analysis module is a relatively small amount has a large volume with a correspondingly low concentration of the target substance.

The object of the invention is to enable improved automated online detection of a biological target substance in a sample liquid and to provide an improved method or an improved device for automated online detection of a biological target substance in a sample liquid. In particular sol The method and the device enable efficient concentration of the target substance and low-loss forwarding of the sample liquid with the concentrated target substance to a subsequent detection unit.

This task is solved by the method for automated online detection of at least one biological target substance in a sample liquid using an online analysis device according to claim 1, by the concentrator unit for automatically carrying out a concentration of a target substance in a sample liquid according to claim 14 and by the online -Analysis device for detecting at least one biological target substance in a sample liquid according to claim 15. Advantageous embodiments are specified in the dependent claims.

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

Superabsorbent polymers (SAP) are plastics that are able to absorb polar liquids many times their own weight. These are primarily water or aqueous solutions. When the liquid is absorbed, the superabsorbent swells and forms a hydrogel. Hydrogels can form any cross-linked polymer that is polar (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) on the one hand and acrylamide on the other hand is usually used, although the ratio of the two monomers to one another can vary. In addition, a so-called core cross-linker (CXL) is added to the monomer solution, which connects the long-chain polymer molecules formed to one another in places using 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 so-called surface cross-linking, called surface cross-linking (SXL). Another chemical is applied to the surface of the particles, which, when heated, forms a second network only on the outer layer of the grain. This cover supports the swollen gel to hold it together even under external stress (movement, pressure).

For example, the product is conventionally used as white granules with particle sizes of 100 to 1000 µm. It is largely used in baby diapers, sanitary napkins, incontinence care, in bandages and, in small quantities, in cable sheathing for deep-sea pipelines. Other areas of application include so-called gel beds, gel-forming extinguishing agents in firefighting, as a mechanical stabilizer for cut flowers in a vase or as an additive to plant soil to permanently store water. However, acrylic acid neutralized with potassium hydroxide is used because of 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 (submillimeter to centimeter).

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 liquid volume of 1 liter. These water beads are made from a super absorbent material. The liquid was surface water that had been taken from a fire-fighting water 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 reduced. Surprisingly, it was found that the suspended particles in the liquid were not absorbed by the swelling beads. The liquid portion of the mixture including the suspended matter (volume 400 ml) was transferred to a new vessel. A sample with a volume of 50 ml was taken from this liquid portion.

A comparison sample with a volume of 50 ml was taken directly from the liquid, i.e. the surface water mentioned, without first concentrating the liquid using the method described. Both samples were centrifuged at 5000 × g for 10 min. The supernatant was removed and the sediment used for nucleic acid extraction. Nucleic acid extraction was carried out using a commercial kit (innuprep Stool DNA Mini Kit; IST Innuscreen GmbH). The DNA from both samples was then examined for the amount of total bacterial count using real time PCR.

This showed that fewer bacteria were detected in the untreated comparison sample than in the sample concentrated with the superabsorbent. This means that the bacteria contained in the liquid has not been absorbed into the water beads. They were confiscated centered and were part of the remaining liquid portion of the mixture of the liquid and the superabsorbent balls. These observations were subsequently confirmed for other biomolecules (viruses or eukaryotic cells): After adding the superabsorbent to a volume of liquid containing these biomolecules, the volume was narrowed down and the biomolecules were concentrated in the remaining liquid. What was even more surprising was that the method described can also be used to concentrate proteins and free genomic DNA that are found in low concentrations in an aqueous solution.

On the basis of this observation, the problem on which the invention is based could be solved.

• - - Erzeugen von aufkonzentrierter Probeflüssigkeit aus mindestens einem ersten initialen Volumen der Probeflüssigkeit mittels:
  1. a. Dosierens und Transportierens eines ersten initialen Volumens der Probeflüssigkeit über eine Flüssigkeitszuleitung in eine erste Konzentratorkammer einer Konzentrator-Einheit; und
  2. b. Reduzierens des ersten initialen Volumens der Probeflüssigkeit in der Konzentrator-Einheit und damit verbundenem Aufkonzentrieren der im ersten Volumen der Probeflüssigkeit enthaltenen mindestens einen biologischen Zielsubstanz mittels einer ersten Menge eines Superabsorbers, wobei das Reduzieren und das damit verbundene Aufkonzentrieren durch Inkubieren eines aus dem ersten initialen Volumen der Probeflüssigkeit mit der ersten Menge des Superabsorbers (24) gebildeten Gemisches erfolgt;
• - Transportieren mindestens eines Teils der erzeugten aufkonzentrierten Probeflüssigkeit aus der Konzentrator-Einheit (3) in eine Detektionseinheit des Online-Analysegeräts.

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

• - - Generating concentrated sample liquid from at least a first initial volume of the sample liquid by means of:
  1. 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
  2. b. Reducing the first initial volume of the sample liquid in the concentrator unit and associated concentration of 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, the reduction and the associated concentration by incubating one of the first initial volume the sample liquid is mixed with the first amount of the superabsorbent (24);
• - Transporting at least part of the concentrated sample liquid produced from the concentrator unit (3) into a detection unit of the online analysis device.

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

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 method can be compared to the method described in the prior art in which biomolecules are released after binding to a filter medium by rinsing out the filter medium, a comparatively high proportion of the biomolecules obtained from the first initial volume of the sample liquid are concentrated in a small volume of liquid. 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 keep the volume intended for the subsequent analysis, for example to be removed from a process, comparatively small, even with a low concentration of the target substance in the sample liquid to be examined to choose. This makes the method universally suitable for monitoring liquids in a variety of different industrial processes, regardless of whether the volume of sample liquid transported into the process unit has to be discarded after enrichment and, if necessary, disposed of, or whether it can be returned to the process .

The target substance can be, for example, a biomolecule. The biomolecule can be selected from the group formed by: 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 used as a sample liquid, for example water and wastewater samples, fermentation liquids, liquids from food technology, chemical, pharmaceutical or biochemical production processes.

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

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 process and/or by detecting metabolic and/or enzymatic activity of microorganisms.

The sample liquid concentrated using superabsorbers using the method according to the invention can therefore be used in a variety of detection and analysis methods. It is clear to the person skilled in the art that the concentrated sample liquid can be examined in a variety of other detection and analysis methods not listed here.

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 auto-fluorescence measurement or by means of a fluorescent label.

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 using a combination of fluorescence measurement and particle scattering.

Using both configurations, a statement can be made about the amount of biological target substance present in the sample.

• - Ein Sensor basierend auf einer zeitaufgelösten Fluoreszenzmessung, wobei die Hintergrundfluoreszenz durch ein Time-Gate (Zeitgatter) entfernt wird;
• - Ein Sensor basierend auf Ramanspektroskopie;
• - elektrochemischer Sensor
• - ein Sensor mit einer aktivierten Oberfläche, welche Oberfläche das Anbinden von Biomolekülen erlaubt, wobei die Anbindung optisch oder elektrisch erfasst werden kann;
• - ein Farbumschlagssensor;
• - ein Sensor basierend auf Absorptionsspektroskopie.

When using a sensor, the sensor can be designed based on a wide variety of physical and/or chemical measurement principles, comprising:

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

If 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.

According to an advantageous embodiment of the method according to the invention, it is provided that if the qualitative or quantitative determination is carried out using a nucleic acid hybridization method, labeled probes are used. Common methods such as in situ hybridization can be used to detect RNA or DNA sequences.

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

For example, it is envisaged that the measurement of enzymatic activity is carried out by measuring the beta-D-glucuronidase activity in the event that a sample is to be examined for coliform germs as microorganisms. Coliform germs, 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 β-glucuronidase, abbreviation GUS) is a bacterial enzyme that catalyzes the hydrolysis of glycosides of glucuronic acid (glucuronides). Based on the cleavage products formed during this reaction (or the amount produced), a statement can be made about the enzymatic activity and, derived from this, the presence and even the amount of microorganisms.

Producing concentrated sample liquid can include metering the first initial volume of the sample liquid to the first amount of superabsorbent presented in the first concentrator chamber or metering the first amount of superabsorbent to the first initial volume of sample liquid presented in the first concentrator chamber.

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

The concentrator chamber and the mixture contained therein can be tempered during incubation using a temperature control device in the concentrator unit. The temperature control device can be a heating and/or cooling device that can be operated automatically by means of control electronics for controlling and/or regulating a temperature of the mixture present in the concentrator chamber.

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 analysis device. Alternatively, as described below, the concentrated sample liquid can first be fed to a collecting vessel. This makes it possible to concentrate the target substance in several initial liquid volumes, for example taken as samples from a process or a body of water, using the method described and to combine them for further detection. The combined concentrated samples can optionally be further concentrated again using the method described. This procedure is particularly advantageous if the concentration of the target substance in the original, untreated sample liquid is particularly low. The procedure is described in more detail below.

The sample liquid taken from the concentrator chamber and concentrated in a first stage can first be transported into a collecting vessel. Subsequently, residues of the concentrated sample liquid and the first amount of superabsorbent can be removed from the concentrator chamber, for example by flushing the concentrator chamber with a flushing fluid, for example a flushing liquid or a flushing gas.

• - Dosieren und Transportieren eines zweiten initialen Volumens der Probeflüssigkeit über eine Flüssigkeitszuleitung in die erste Konzentratorkammer der Konzentrator-Einheit; und
• - Erzeugen von aufkonzentrierter Probeflüssigkeit durch Reduzieren des zweiten initialen Volumens der Probeflüssigkeit in der Konzentrator-Einheit und damit verbundenem Aufkonzentrieren von in dem zweiten initialen Volumen der Probeflüssigkeit enthaltenen Biomolekülen mittels einer zweiten Menge des Superabsorbers, wobei das Reduzieren und das damit verbundene Aufkonzentrieren durch Inkubieren eines aus dem zweiten initialen Volumen der Probeflüssigkeit mit der zweiten Menge des Superabsorbers gebildeten Gemisches erfolgt, und
• - Transportieren mindestens eines Teils der durch Reduzieren des zweiten initialen Volumens der Probeflüssigkeit erhaltenen aufkonzentrierten Probeflüssigkeit aus der ersten Konzentratorkammer in das Sammelgefäß.

The process can further include the following steps:

• - 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
• - Generating concentrated sample liquid by reducing the second initial volume of the sample liquid in the concentrator unit and associated concentration of biomolecules contained in the second initial volume of the sample liquid by means of a second amount of the superabsorbent, the reduction and the associated concentration by incubating a The mixture formed from the second initial volume of the sample liquid with the second amount of the superabsorbent takes place, and
• - Transporting at least a portion of the concentrated sample liquid obtained by reducing the second initial volume of the sample liquid from the first concentrator chamber into the collecting vessel.

In this way, several batches of concentrated sample liquid can be produced and combined in the collection vessel. This makes it possible to concentrate a very large volume of sample.

At least part of the concentrated sample liquid from the collecting vessel can be metered and transported into the first concentrator chamber or into a second concentrator chamber that is different from the first concentrator chamber for further concentration of the target substance using a third amount of the superabsorbent. This further concentration is carried out in a completely analogous manner to that described for the first concentration, namely by producing a mixture of the concentrated sample liquid and the third amount of the superabsorbent and incubating the mixture, whereby the volume of the concentrated sample liquid is reduced again and the biomolecules contained therein, including the target substance, needs to be further concentrated. The further concentrated sample liquid obtained in this way can be concentrated again in the same way or fed directly to a detection unit of an analysis device for further analysis. In this way, the sensitivity of the method can be further increased.

The method can advantageously be used in all of its variants described above be carried out completely automatically by control electronics. For this purpose, the concentrator unit can have means for transporting liquids and for transporting the superabsorbent within the concentrator unit, which can include, for example, pumps and/or valves.

• - Freisetzen und/oder Isolieren von Nukleinsäuren aus den in der aufkonzentrierten Probeflüssigkeit enthaltenen Biomolekülen, insbesondere der mindestens einen biologischen Zielsubstanz, und Erzeugen einer die freigesetzten und/oder isolierten Nukleinsäuren umfassenden Lösung,
• - Amplifizieren mindestens einer in der Lösung enthaltenen Ziel-Nukleinsäure, und
• - Erfassen eines Messsignals, das eine von dem Fortschritt der Amplifizierung und/oder von einer Anzahl von Kopien der Ziel-Nukleinsäure abhängige Messgröße repräsentiert, wobei die Steuerelektronik des Analysegeräts anhand des erfassten Messsignals die qualitative oder quantitative Bestimmung der Zielsubstanz in der aufkonzentrierten Probeflüssigkeit und/oder in der Probeflüssigkeit vor Aufkonzentrierung durchführt. Die zu amplifizierende Ziel-Nukleinsäure kann entweder die zu detektierende Zielsubstanz oder eine Nukleinsäure der zu detektierenden Zielsubstanz sein.

The qualitative or quantitative determination can also be carried out using amplification. This is intended for:

• - 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,
• - Amplify at least one target nucleic acid contained in the solution, and
• - Acquiring a measurement signal which represents a measurement variable dependent on the progress of the amplification and/or on a number of copies of the target nucleic acid, the control electronics of the analysis device using the captured measurement signal to determine the qualitative or quantitative determination of the target substance in the concentrated sample liquid and/or or in the sample liquid before concentration. The target nucleic acid to be amplified can either be the target substance to be detected or a nucleic acid of the target substance to be detected.

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 is carried out in a first microfluidic unit of the detection unit. The solution is transported into a second microfluidic unit that can be fluidically connected to the first microfluidic unit, and the amplifying and recording of the measurement signal is carried out in the second microfluidic unit. The first and second microfluidic units can be accommodated in two separate cartridges or in a common cartridge.

• - Versetzen der flüssigen Probe mit einem oder mehreren Lysereagenzien, um Nukleinsäuren der Biomoleküle freizusetzen,
• - Binden der freigesetzten Nukleinsäuren an ein Nukleinsäure bindendes Material; und
• - Waschen der gebundenen Nukleinsäuren mit einer Waschlösung, und
• - Ablösen der Nukleinsäuren von dem Nukleinsäure bindenden Material durch Eluieren.

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 can include at least the following steps:

• - Adding one or more lysis reagents to the liquid sample in order to release nucleic acids of the biomolecules,
• - Binding the released nucleic acids to a nucleic acid-binding material; and
• - Washing the bound nucleic acids with a washing solution, and
• - Detachment of the nucleic acids from the nucleic acid-binding material by elution.

The eluate obtained here forms the solution mentioned, which includes the released and/or isolated nucleic acids.

• - thermisches Behandeln der in der aufkonzentrierten Probeflüssigkeit enthaltenen Biomoleküle in einer Pufferlösung, beispielsweise in einer phosphatgepufferten Salzlösung, in Wasser, in einem Tris-Puffer oder in einem Puffer, der eine geringe Konzentration an einem Detergenz enthält. In diesem Fall bildet die Pufferlösung mit den durch die thermische Behandlung freigesetzten Nukleinsäuren die zuvor erwähnte, die freigesetzten und/oder isolierten Nukleinsäuren umfassende Lösung.

Releasing and/or isolating nucleic acids from the biomolecules contained in the concentrated sample liquid and generating the solution comprising the released and/or isolated nucleic acids can include the following steps:

• - thermally treating the biomolecules contained in the concentrated sample liquid in a buffer solution, for example in a phosphate-buffered saline solution, in water, in a Tris buffer or in a buffer that contains a low concentration of a detergent. In this case, the buffer solution with the nucleic acids released by the thermal treatment forms the aforementioned solution comprising the released and/or isolated nucleic acids.

Thermal release may include dispersing the sample in the buffered solution and incubation. This method variant is suitable, for example, for applications in which the target substance to be determined is a nucleic acid that is already freely present in the liquid to be analyzed. Classic lysis can therefore be omitted. It is also possible to use the method if the target substance as a biomolecule 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.

The amplification carried out, for example, in the second microfluidic unit can be carried out, for example, using conventional PCR-based methods. The qualitative or quantitative determination of the target substance can be carried out, also 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 times, which corresponds to the number present at the respective time through the amplification generated copies of a target nucleic acid are correlated. From the measured value or measured value curve obtained as a function of time, the presence of the target nucleic acid and thus the biological target substance in the sample liquid can be qualitatively detected or determine a quantitative value, for example a concentration, of the biological target substance in the original sample liquid or in the sample liquid after concentration using the method described above and output it as a measurement result by the control electronics.

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

• - eine erste Konzentratorkammer;
• - mindestens eine in die erste Konzentratorkammer mündende Flüssigkeitszuleitung; und
• - mindestens eine in die erste Konzentratorkammer mündende Flüssigkeitsableitung, wobei die Konzentrator-Einheit dazu eingerichtet ist, eine erste Menge eines Superabsorbers zu einem über die Flüssigkeitszuleitung in die erste Konzentratorkammer eingeleiteten initialen Flüssigkeitsvolumen der Probeflüssigkeit zuzugeben, oder dazu eingerichtet ist, das initiale Flüssigkeitsvolumen zu der ersten Menge des Superabsorbers zuzugeben.

The invention also includes a concentrator unit for automatically carrying out a concentration of a target substance in a sample liquid, in particular after the first method step of the method described above, comprising:

• - a first concentrator chamber;
• - at least one liquid supply line opening into the first concentrator chamber; and
• - at least one liquid discharge line opening into the first concentrator chamber, wherein the concentrator unit is set up 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 set up to add the initial liquid volume to the Add the first amount of superabsorbent.

The first amount of superabsorbent can be placed in the first concentrator chamber. The concentrator chamber can be designed as a replaceable cartridge. In order to concentrate the target substance in each new batch of sample liquid, the replaceable cartridge with the superabsorbent can be replaced with a new cartridge.

Alternatively, the concentrator unit can have at least one reservoir or be connected to a reservoir in which superabsorbent, in particular in the form of particles, is contained. One of the materials mentioned above, in particular in spherical shape, is preferably used as the superabsorber. Commercially available superabsorbent beads with a diameter of around 1 mm, which are also commercially available under the names “water beads”, “aqua beads”, “water beads” or “gel beads”, have proven to be very advantageous. Superabsorber spheres of this size can be dispersed very well in the concentrator chamber.

The reservoir can, for example, be connected to the first concentrator chamber via a superabsorber feed line that can be closed with a valve, in particular controllable by means of control electronics, such that when the valve is open, at least part of the superabsorbent contained in the reservoir is released into the first amount of superabsorbent as the first amount of superabsorbent concentrator chamber. The transport of superabsorbents can be achieved using gravity. However, it is also possible for the concentrator unit to have means for transporting the superabsorbent, for example present in particles, for example a pump and/or means for generating a gas stream which transports the superabsorbent particles. For dosing and transporting the superabsorbent, control electronics designed for automatic operation of the concentrator unit can be used, which is set up to actuate the mentioned means and/or the mentioned valve in order to measure the first amount of superabsorbent and transport it into the concentrator chamber. This control electronics can be set up in conjunction with pumps and/or valves to meter the initial volume of the sample liquid into the concentrator chamber.

The concentrator device can have means for moving, in particular stirring, the liquid contained in the reaction chamber. The means can, for example, include a stirrer, a feed line for an inert gas into the reaction chamber and a discharge line for the inert gas, which are arranged so that the inert gas flows through the liquid contained in the reaction chamber, or have a drive for moving the reaction chamber.

The concentrator unit can further have a temperature control device which is designed to control the temperature of the first concentrator chamber. The temperature control device can, for example, have heating and/or cooling elements that can be operated by means of the control electronics in order to control and/or regulate a temperature of a liquid held in the concentrator chamber.

The liquid discharge for discharging at least a portion of the liquid volume of the product present in the first concentrator chamber fluid can be fluidly connectable to a collecting vessel.

The collecting vessel can have a collecting vessel drain that can be fluidly connected to the first concentrator chamber. In this way, sample liquid present in the collecting vessel that has already been concentrated in a first stage can be fed back into the first concentrator chamber in order to be further concentrated in a second stage by again feeding superabsorbent to the concentrated sample liquid in the first concentrator chamber and incubating the mixture.

Alternatively, the concentrator unit can comprise an additional second concentrator chamber, the liquid discharge of the first concentrator chamber connecting 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 set up to add a second amount of the superabsorbent to a volume of 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.

The invention also includes 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 includes: control electronics;
a concentrator unit according to one of the embodiments described above, 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 that can be fluidly connected to the liquid outlet of the concentrator unit; and at least one transport device that can be controlled by the control electronics, for example a pump, which is set up to transport concentrated sample liquid from the liquid outlet of the concentrator unit into the detection unit, the control electronics being set up to do this by means of the

Detection unit 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.

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, which are required for an immunological detection, via separate inlet openings

According to an advantageous embodiment of the online analysis device according to the invention, it is provided that, in order to carry out the measurement of the amount of adenosine triphosphate present in the sample liquid, the detection unit is designed in such a way that it allows the supply 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 any dysfunction of the mitochondria. For example, ATP can be rapidly detected by light emission through the combined use of luciferase and a luminometer.

According to an advantageous embodiment of the online analysis device according to the invention, it is provided that if a nucleic acid hybridization process is carried out using labeled probes, the detection unit has separate inlet openings which allow the supply of reagents that are required for the nucleic acid hybridization process . Common reagents for this are primers, nucleotides, buffers, etc.

According to an advantageous embodiment of the online analysis device according to the invention, it is provided that if the measurement of enzymatic activity takes place 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 measuring beta-d-glucuronidase activity.

The online analysis device can also have a sample pump, with the control electronics being set up to control the sample pump to transport a predetermined volume of sample liquid into the concentrator unit. The sample supply line can be fluidly connectable to a sample template or directly to a body of water or a line in a process plant, a fermenter or another process container.

• - eine mit einem Flüssigkeitsausgang für aufkonzentrierte Probenflüssigkeit der Konzentrator-Einheit fluidisch verbindbare erste Mikrofluidikeinheit, die dazu eingerichtet ist, aus in der aufkonzentrierten Probeflüssigkeit enthaltenen Biomolekülen, insbesondere der mindestens einen biologischen Zielsubstanz, Nukleinsäuren freizusetzen und/oder zu isolieren,
• - eine mit der ersten Mikrofluidikeinheit verbundene zweite Mikrofluidikeinheit, die dazu eingerichtet ist, ein die freigesetzten und/oder isolierten Nukleinsäuren umfassendes Eluat aufzunehmen und eine Ziel-Nukleinsäure zu amplifizieren; und
• - einen, insbesondere optischen, Sensor, der dazu eingerichtet ist, ein von dem Fortschritt einer in der zweiten Mikrofluidikeinheit durchgeführten Amplifizierung und/oder von einer Anzahl von Kopien der Ziel-Nukleinsäure in der zweiten Mikrofluidikeinheit abhängiges Messsignal zu erzeugen und an die Steuerelektronik auszugeben; und

wobei die Steuerelektronik dazu eingerichtet ist, anhand des von dem Sensor ausgegebenen Messsignals die biologische Zielsubstanz in der Flüssigkeit qualitativ und/oder quantitativ zu bestimmen.The detection unit can have the following components:

• - A first microfluid that can be fluidically connected to a liquid outlet for concentrated sample liquid of the concentrator unit dic unit which is designed 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 designed 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 set up to generate a measurement signal which is dependent on the progress of an amplification carried out in the second microfluidic unit and/or on a number of copies of the target nucleic acid in the second microfluidic unit and output it to the control electronics; and

wherein the control electronics is set up to qualitatively and/or quantitatively determine the biological target substance in the liquid based on the measurement signal output by the sensor.

The liquid outlet of the concentrator unit can be fluidly connected to the aforementioned first concentrator chamber and/or to an optionally present second concentrator chamber and/or to a collecting vessel which is also optionally present. In various design or operating alternatives of the online analysis device, concentrated sample liquid can thus be transported directly from the first or the optionally available second concentrator chamber or via the optionally available collecting vessel into the detection unit.

The first and second microfluidic units can be arranged together in a, in particular replaceable, cartridge. Alternatively, the first microfluidic unit can be arranged in a first cartridge and the second microfluidic unit can be arranged in a second cartridge that is different from the first cartridge, wherein the first and second cartridges can be fluidly connected to one another in order to transport liquid from the first cartridge into the second . The first and second cartridges can be designed to be interchangeable.

The first microfluidic unit can be set up to receive the concentrated sample with the biomolecules contained therein and to add one or more lysis reagents in order to release nucleic acid of the biomolecules contained in the sample, then the released nucleic acids, if necessary with the addition of a binding of the nucleic acid to a To bind nucleic acid-binding material reinforcing/mediating component and to wash the nucleic acids bound to this material once or several times with a washing solution. The control electronics can be set up to control transport of the particles and optionally the reagents through the first microfluidic unit.

The first microfluidic unit can further be set up to detach 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 set up to control the elution and transport of the eluate.

Alternatively, the first microfluidic unit can be set up to receive the concentrated sample with the biomolecules contained and to store 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 contains, thermally/chemically released, the control electronics being set up to control the thermal release of the biomolecules and to transport the buffer solution with the biomolecules or nucleic acids dissolved therein into 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.

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 closed by microvalves or in reagent packs closed by foils, which are automated by the action of force or heat. in particular by means of the control electronics.

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

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 have 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 means of the processor, to the analysis device to control automatically and to determine qualitative or quantitative analysis results from the measurement signals from the sensor. The operating programs can be designed so that the control electronics controls the analysis device to carry out the method described above. The control electronics can also have a user interface, for example a touch screen or another display in combination with an input keyboard. The control electronics can be set up to be connected wirelessly by radio or via a data line to another data processing device, for example a computer, a process control, a display or operating device, in particular a portable one, for communication.

The automatic control of the concentration carried out in the concentrator unit can be carried out using the same control electronics that are also used to control the other components of the analysis device. Alternatively, the concentrator unit can also have its own control electronics, which is set up to automatically carry out the concentration of the sample liquid in the manner described. The control electronics of the concentrator unit are here advantageously connected for communication with the control electronics of the analysis device, which controls the remaining components of the analysis device, namely in particular the detection unit.

The analyzer can thus perform online determinations of biological target substances in a liquid without requiring manual sampling or preparation or transport of samples to a laboratory. In particular, the analysis device can carry out sampling and detection cycles completely automatically, which can, for example, be event-controlled by the control electronics or carried out at predetermined time intervals.

• 1 eine schematische Darstellung eine Online-Analysegeräts zur qualitativen oder quantitativen Bestimmung einer biologischen Zielsubstanz in einer Flüssigkeit;
• 2 ein erstes Ausführungsbeispiel einer Konzentrator-Einheit des in 1 dargestellten Online-Analysegeräts nach einem ersten Ausführungsbeispiel; und
• 3 ein zweites Ausführungsbeispiel einer Konzentrator-Einheit für ein Online-Analysegerät.

The invention is described below using the exemplary embodiments shown in the figures. The same reference numbers designate the same components of the components shown in the figures. Show it:

• 1 a schematic representation of an online analysis device for the qualitative or quantitative determination of a biological target substance in a liquid;
• 2 a first embodiment of a concentrator unit in 1 online analysis device shown according to a first exemplary embodiment; and
• 3 a second embodiment of a concentrator unit for an online analysis device.

In 1 An exemplary embodiment of an online analysis device 1 for detecting a biological target substance in a sample liquid is shown schematically. In the present example, the analysis device 1 has a housing 2 in which all components of the device are accommodated. These components include in detail 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 in very low concentrations, since it is set up to detect biological target molecules in a volume of a to concentrate the sample liquid to be analyzed and to pass on the concentrated sample liquid to the detection unit 4 for detection and, if necessary, preparatory steps such as extraction and / or isolation of nucleic acids.

The concentrator unit 3 is fluidly connected via a sample feed line 6 to a sample template 7 or to a process container, e.g. a pipeline or 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 template 7 into a first concentrator chamber 8 contained in the concentrator unit 3 and can be controlled by the control electronics 5 according to an operating program stored in the control electronics 5 and executed by it. The first concentrator chamber 8 can also be fluidly connected to reservoirs 10, 11. The reservoirs 10, 11 contain substances that serve to enrich biomolecules contained in the liquid. In the present example, each reservoir 10, 11 is fluidly 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 substances can of course be transported using other means known to those skilled in the art, for example pneumatically or by using gravity or, for example, hydrostatic pressure.

The concentrator unit 3 can optionally have a temperature control unit (not in 1 shown), which is used to set a specific temperature in the concentrator chamber 8. The tem Perier unit may include resistance heating, cooling and/or thermoelectric elements for selective heating or cooling. It can be connected to the control electronics 5, the control electronics 5 being set up to control or regulate the temperature in the concentrator chamber 8.

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.

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

In the present example, the detection unit 4 has an analysis cartridge in which two microfluidic units fluidly connected to one another via the fluid line 18, namely a first microfluidic unit 19 and a second microfluidic unit 20, are integrated. In another embodiment, the microfluidic units can also be accommodated in separate cartridges. The analysis cartridge is replaceable in this example.

The first microfluidic unit 19 is set up 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 processes. For this purpose, the first microfluidic unit 19 includes reagents and optionally a temperature control unit for incubating reaction mixtures produced in the microfluidic unit 19. To control 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 can be controlled by the control electronics 5. The second microfluidic unit 20 includes means for amplification, i.e. reagents and temperature control elements, for example thermoelectric elements, as well as means for transporting and aliquoting fluids within the second microfluidic unit 20. To detect progress of the amplification in a manner known per se, for example by means of real time PCR-based method, the detection unit has a sensor 21, which can, for example, carry out fluorescence measurements in the second microfluidic unit 20 and can output measurement signals to the control electronics 5. The control electronics 5 is set up to determine a qualitative or quantitative analysis result based on the measurement signals.

The control electronics 5 can be a central processing unit, for example a CPU with a processor and memory and operating programs stored therein. It can also be divided into several computing units within the analysis device, for example the concentrator unit 3 and the detection unit 4 can each have their own on-site electronics, which are connected to higher-level electronics for communication, the higher-level electronics and the on-site electronics together form the control electronics 5.

In 2 is a schematic of a first exemplary embodiment of a concentrator unit 3 of FIG 1 online analysis device 1 shown in detail. The in 2 The structure of the concentrator unit 3 shown is merely a possible exemplary embodiment. The person skilled in the art can find a large number of variants without deviating from the idea of the invention.

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 superabsorber 24, for example in the form of balls made of the superabsorbent material. The superabsorber 24 is added to the concentrator chamber 8 via the supply line 23. A first opening 25 is used to supply the sample liquid. This is used to enter a specific initial volume of the sample liquid into the concentrator chamber 8. A second opening 26 is connected to the fluid line, which connects 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 superabsorber spheres, so that only the liquid portion of the mixture of sample liquid and adsorber can be derived from the concentrator chamber 8 via the second opening 26 in the direction of the detection unit 4. Optionally, the second opening 26 can also have a size selector, for example a grid or a filter, which retains the superabsorber balls in the concentrator chamber 8.

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 that serves as a discharge, which in the present example can be introduced into the concentrator chamber 8 opposite the opening 25 via the sample liquid. To completely remove the superabsorber 24 and the remaining sample liquid A flushing liquid or a flushing 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 template 7 through the concentrator chamber 8. Alternatively, the flushing liquid can be a liquid stored in the reservoir 11. In the latter case, the reservoir 11 is fluidly connected to the first opening 25. Optionally, valves are arranged in the respective supply lines and can be actuated by the control electronics 5.

• 1) Einleiten eines ersten initialen Probeflüssigkeitsvolumens aus der Probevorlage 7 in die Konzentratorkammer 8,
• 2) Zugeben einer ersten Menge des Superabsorbers 24 aus dem Reservoir 10 in die Konzentratorkammer 8 zu dem ersten initialen Probeflüssigkeitsvolumen,
• 3) Inkubieren des Gemisches aus dem Superabsorber 24 und der Probeflüssigkeit während einer von der Steuerelektronik 5 gemessenen Zeitspanne, dabei reduziert sich das Volumen des flüssigen Anteils des Gemisches unter Aufkonzentrierung von Biomolekülen, insbesondere einer zu bestimmenden biologischen Zielsubstanz in dem flüssigen Anteil,
• 4) Nach Ablauf der Zeitspanne für die Inkubation Ableiten des flüssigen Anteils.

The control electronics 5 can include an operating program that it can execute to carry out the following method:

• 1) introducing a first initial sample liquid volume from the sample template 7 into the concentrator chamber 8,
• 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,
• 3) incubating the mixture of the superabsorber 24 and the sample liquid for a period of time measured by the control electronics 5, thereby reducing the volume of the liquid portion of the mixture while concentrating biomolecules, in particular a biological target substance to be determined, in the liquid portion,
• 4) After the incubation period has elapsed, drain the liquid portion.

As already described above, the superabsorber absorbs polar solvents, 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 superabsorber. During incubation, the volume of the liquid portion of the mixture of superabsorber and sample liquid decreases, and the target substance is concentrated in the liquid. The incubation time can be chosen so that it is ensured that the concentration of the target substance in the concentrated sample liquid after incubation is above a threshold value, e.g. above a detection limit specified by the detection unit. The time until the desired volume reduction or the desired concentration depends on the following factors: type of superabsorbent, particle size of the superabsorbent particles, amount of superabsorbent added, temperature during incubation. A suitable amount of superabsorbent and incubation time for a specific type of sample liquid can be determined using preliminary tests and stored in the control electronics.

The control unit 5 can be designed to set or regulate the temperature of the liquid or the mixture of sample liquid and superabsorber contained in the concentrator chamber 8 to a constant value by means of the aforementioned temperature control device. All of these steps can be carried out 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 superabsorber. In a possible embodiment according to the exemplary embodiment described here, the concentrated sample liquid can be transported directly into the detection unit 4. With the method described here and the concentrator unit 3 described here for carrying out this method, it becomes possible to reduce a large-volume water sample to a much smaller sample volume and, correspondingly, to achieve a large 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 originally very low, the method described here can even enable molecular genetic detection of the target substance.

A further advantage arises from the fact that with the approach according to the invention it is possible to concentrate both bacteria, viruses and free nucleic acids in a completely non-selective manner. Accordingly, it is also possible in a very simple manner to initially concentrate, extract and then qualitatively or quantitatively determine several different biological target substances using the method. For example, the qualitative or quantitative determination of the biological target substance present in the concentrated sample liquid is carried out in the detection unit 4 by means of a sensor, by means of an immunological detection method, by measuring an 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 or by recording the metabolic and/or enzymatic activity of microorganisms.

The opening 26 of the concentrator unit 3 is fluidly connected to the fluid line 15 of the analysis device 1, which can be connected via the valve 16 on the one hand to the detection unit 4 and on the other hand to the liquid outlet 17.

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

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 acids already present in the liquid taken from the sample template, the biomolecules contained in the concentrated sample liquid are lysed. The free nucleic acids are bound to a material that binds nucleic acids, for example in particle form. The nucleic acid-binding material with the nucleic acids bound to it is subsequently washed in a known manner and the nucleic acids are finally dissolved from the material. The eluate containing the dissolved nucleic acids is transported into the second microfluidic unit 20, again controlled by the control electronics 5.

In the second microfluidic unit 20, certain volumes of the eluate are transported into 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 means of fluorescence measurements with the sensor 21. The sensor 21 outputs measurement signals to the control electronics 5. This determines from the measurement signals either a qualitative value that indicates whether the specific biological target substance is contained in the liquid taken from the sample template 7. Alternatively or additionally, the control electronics 5 can also determine a quantitative value from the measurement signals, which 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 and thus several different biological target substances in the liquid can be qualitatively or quantitatively determined in parallel.

Before or after the transfer of the concentrated liquid from the concentrator unit 3 into the detection unit 4 or after detection has taken place, the liquid remaining in the concentrator unit 3 is removed 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 sucked in from the sample template 7, and the process can now be repeated for another measurement. The microfluidic units 19, 20 in the detection unit can be replaced by new microfluidic units 19, 20 for further measurement. All steps described here can be carried out automatically by the control electronics 5.

In 3 An alternative embodiment of a concentrator unit 33 is shown schematically, which is used instead of the one in 2 Concentrator unit 3 shown in the analysis device 1 can be used. The concentrator unit 33 is set up to concentrate a sample liquid in several stages and thus allows the concentration of even target substances that are only present in 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 superabsorbents, for example in the form of balls with a diameter of the order of 1 mm, and two discharge 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 drain 26 from the concentrator chamber 8 leads to a collecting vessel 28, the second drain 27 serves as a drain for used liquid and used superabsorbent and can be connected, for example, to a waste collection container.

The collecting vessel 28 can be fluidly connected to a second concentrator chamber 30 via a liquid discharge line 29. The second concentrator chamber 30 can be designed identically to the first concentrator chamber 8. In the present example, it has a further supply line 31 for superabsorbents, which can be fluidly connected to the same reservoir 10 of the analysis device as the supply line 23 to the first concentrator chamber 8. The second concentrator chamber has a first lead device 15, which can be fluidly connected to the detector unit 4 of the analysis device in order to supply the detector unit with concentrated sample liquid for the qualitative and/or quantitative detection of the biological target substance. The concentrator chamber 8 has a second discharge line 32 for discharging used liquid and/or used superabsorbent.

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 automatically 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 to sample liquid to be analyzed. After adding a first 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 part of the liquid portion remaining after incubation can then be passed via the first discharge line 26 from the concentrator chamber 8 into the collecting vessel 28. Residual liquid and used superabsorbent can be drained from the concentrator chamber via drain 27.

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

The collected sample liquid, which has already been concentrated in a first stage, can be further concentrated in a second stage using the second concentrator chamber 30. For this purpose, at least part of the concentrated sample liquid contained in the collecting vessel 28 can be introduced into the second concentrator chamber 30 via the drain 29 of the collecting vessel 28. A second amount of superabsorber is introduced into the second concentrator chamber 30 via the supply line 31 for superabsorbents. 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 part of the liquid portion can then be supplied to the detector unit 4 of the analysis device via the liquid line 15. Unnecessary liquid and/or the used superabsorbent can be drained from the second concentrator chamber 30 via the drain line 32.

All steps of the method described here can be carried out completely automatically by control electronics, for example the control electronics 5 of the analysis device 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 in 3 shown), which actuates the control electronics to carry out the described method for concentrating the target substance in the sample liquid.

The analysis device and method according to the invention thus ideally solves the task by linking available device systems for detection and enables online liquid analysis of biological targets to be carried out, which is particularly advantageous for monitoring water or wastewater in networks, sewage treatment plants or treatment plants as well as from 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 economical manner without manual intervention, starting with a large-volume sample and ending with detection.

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 20150224502 A1 [0005, 0006, 0007]
• WO 2013045631 A1 [0094]
• EP 2621632 A1 [0094]

Claims (19)

Method for the automated online detection of at least one biological target substance in a sample liquid using an online analysis device (1), comprising: - Generating concentrated sample liquid from at least a first initial volume of the sample liquid by means of: c. Dosing and transporting 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 d. Reducing 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 contained in the first volume of the sample liquid by means of a first amount of a superabsorbent (24), the reduction and the associated concentration by incubating a mixture formed from the first initial volume of the sample liquid with the first amount of the superabsorbent (24); - transporting at least part of the concentrated sample liquid produced from the concentrator unit (3) into a detection unit (4) of the online analysis device, - 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 measuring an 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 recording the metabolic and/or enzymatic activity of microorganisms. Procedure according to Claim 1 , whereby in the case of a flow cytometric method, the biological target substance present in the concentrated sample liquid is detected by means of an auto-fluorescence measurement or by means of a fluorescent label. 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 using a combination of fluorescence measurement and particle scattering. Procedure according to Claim 1 , where the sensor is based on a time-resolved fluorescence measurement and the background fluorescence is removed by a time gate. Procedure according to Claim 1 , where the sensor is based on Raman spectroscopy. Procedure according to Claim 1 , whereby an electrochemical sensor is used as a sensor. Procedure according to Claim 1 , whereby a sensor with an activated surface is used as a sensor, which surface allows the binding of biomolecules, whereby the binding can be detected optically or electrically. Procedure according to Claim 1 , whereby a color change sensor is used as the sensor. Procedure according to Claim 1 , where the sensor is based on absorption spectroscopy. Procedure according to Claim 9 , whereby an Attenuated Total Reflection (ATR) method, a photothermal method or the transmission spectroscopy method is used for absorption spectroscopy. Procedure according to Claim 1 , whereby labeled probes are used to carry out a nucleic acid hybridization process. Procedure according to Claim 1 , wherein the detection of the biomolecules, preferably bacteria, in the concentrated sample present in the detection unit (4) is carried out by measuring the metabolic or enzymatic activity of microorganisms. Procedure according to Claim 12 , whereby the measurement of enzymatic activity is carried out by measuring the beta-d-glucuronidase activity of coliform germs. Concentrator unit (3, 33) for automatically carrying out a concentration of a target substance in a sample liquid after the first process step of the method according to one of Claims 1 until 13 , comprising: - a first concentrator chamber (8); - at least one liquid supply line (25) opening into the first concentrator chamber (8); and - at least one liquid discharge line (26) opening into the first concentrator chamber (8); wherein the concentrator unit (3, 33) is set up to add a first amount of a superabsorber (24) to an initial liquid volume of the sample liquid introduced into the first concentrator chamber (8) via the liquid supply line (25), or is set up to do so Add initial liquid volume to the first amount of superabsorbent (4). Online analysis device (1) for detecting at least one biological target substance in a sample liquid according to the method according to one of Claims 1 until 13 , comprising: - control electronics (5); - a concentrator unit (3, 33). Claim 14 , wherein the concentrator unit (3, 33) has a liquid outlet for concentrated sample liquid produced from the sample liquid in the concentrator unit (3, 33); and - a detection unit (4) which can be fluidly connected to the liquid outlet of the concentrator unit (3, 33); and - at least one transport device that can be controlled by the control electronics (5), for example a pump, which is set up to transport concentrated sample liquid from the liquid outlet of the concentrator unit (3, 33) into the detection unit (4), - the control electronics (5 ) is set up to use the detection unit (4) 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. Online analyzer Claim 15 , wherein to carry out an immunological detection, the detection unit (4) is designed in such a way that it allows the supply of reagents required for an immunological detection via separate inlet openings Online analyzer Claim 15 , whereby to carry out the measurement of the amount of adenosine triphosphate present in the sample liquid, the detection unit (4) is designed in such a way that it allows the supply of reagents required for the measurement via separate inlet openings. Online analyzer Claim 15 , whereby in the event that a nucleic acid hybridization process is carried out using labeled probes, the detection unit (4) has separate inlet openings which allow reagents required for the nucleic acid hybridization process to be supplied. Online analyzer Claim 15 , whereby in the event that the measurement of enzymatic activity takes place by measuring a beta-d-glucuronidase activity of coliform germs, the detection unit (4) has separate inlet openings which allow the supply of reagents which are necessary for measuring the beta- d-Glucuronidase activity is required.

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