Superabsorbent and kit comprising a superabsorbent

Abstract

Superabsorbent (2) comprising a plastic that absorbs water to form a hydrogel and that absorbs essentially no biomolecules, in particular essentially no eukaryotic cells, components of eukaryotic cells, prokaryotic cells, components of prokaryotic cells, subcellular vesicles, bacteriophages, viruses or virus components, toxins, antibodies, nucleic acids or proteins.

Description

[0001] The invention relates to a superabsorbent. Furthermore, the invention comprises a kit comprising a superabsorbent according to the invention.

[0002] The detection of target substances, especially biological target substances, plays an important role in liquid analysis. Target substances to be detected or monitored can be biomolecules, such as eukaryotic cells, prokaryotic cells, subcellular vesicles, bacteriophages, viruses, toxins, antibodies, or even nucleic acids or proteins. Analytical methods are used for the qualitative or quantitative determination of target substances in samples taken from a liquid to be analyzed. Manual, automated, or at least semi-automated laboratory procedures or fully automated analytical devices are available for this purpose. Automated analytical devices also include online analytical devices that continuously or discontinuously take samples from the liquid to be monitored and perform a qualitative or quantitative determination of the target substance.

[0003] When the concentration of the target substance in the liquid to be analyzed is low, the problem arises of making a sufficient amount of the target substance available for subsequent analysis when taking the sample. In some applications, the target substance is present in an untreated liquid sample at a concentration that is too low for subsequent processing or analysis. This results in the need to concentrate the target substance in the sample. An example of this is the detection of biomolecules in water or wastewater, e.g. the detection of SARS-CoV-2 in water using molecular genetic techniques such as PCR or real-time PCR. The concentrations of the virus particles or viral fragments to be detected in the wastewater are often too low to be detected using methods known to those skilled in the art.While a sample volume of 200 µl is sufficient for testing blood samples for viral infection, the required sample volume for testing water / wastewater is significantly larger. Initial volumes of up to several liters have been described in the literature.

[0004] The concentration of target molecules in a liquid volume plays an important role not only in the preparation of samples for molecular genetic analysis techniques, but also for all immunological technologies and spectroscopic technologies, such as molecular spectroscopy or mass spectroscopy.

[0005] The concentration of target substances, in particular biomolecules, in a liquid volume also plays a role in the production of raw products or products comprising the target substances or biomolecules for further use for research purposes, for industrial purposes or for therapeutic purposes. Various techniques are known in the art for the enrichment of viruses or subcellular particles from a biological sample, e.g., ultracentrifugation techniques or ultrafiltration. These methods are time-consuming and relatively expensive. Alternative methods involve the precipitation of virus particles using polyethylene glycol / sodium chloride followed by centrifugation (Yamamoto et al., Virology 40 (1970) 734; Morandi et al., J. Clin. Microbiol. 36 (1998) 1543-1538). Various mixtures of PEG and sodium chloride are used, and these reagents are mixed with the biological sample. The mixture is then incubated for an extended period in the cold, and the virus (protein)-NaCl / PEG precipitates are subsequently collected by centrifugation. These methods are also complex and time-consuming. Furthermore, the further processing of the precipitates for the isolation of viral nucleic acids is problematic.Often, the precipitates are very difficult to resolubilize. This significantly impacts the efficiency and quality of nucleic acid isolation.

[0006] US 2015 / 0224502 ​​A1 discloses a sample collection device for flow-through sampling in water bodies. To concentrate target substances present in low concentrations in water bodies, the method used is to retain the target substances in a filter or adsorption medium and release the substances adsorbed on the filter for subsequent analysis by elution or as a lysate and make them available to an analysis module. This method is also complex in terms of equipment and not universally applicable.

[0007] The object of the invention is to provide a simple, rapid and universally applicable possibility for producing a sample containing at least one target substance from a volume of a sample liquid containing the at least one target substance for subsequent analysis or as a raw product or product for subsequent use for research purposes, for therapeutic purposes or for industrial purposes, e.g. for product manufacturing.

[0008] This object is achieved in a surprisingly simple and universally applicable manner by means of the superabsorbent defined in claim 1. The object is similarly achieved by means of the kit defined in claim 2. The object is also achieved by the kit defined in claim 3. Advantageous embodiments are specified in the dependent claims.

[0009] The superabsorbent according to the invention comprises a plastic which absorbs water to form a hydrogel and which absorbs essentially no biomolecules, in particular essentially no eukaryotic cells, components of eukaryotic cells, prokaryotic cells, components of prokaryotic cells, subcellular vesicles, bacteriophages, viruses or virus components, toxins, antibodies, nucleic acids or proteins.

[0010] The kit according to the invention is designed to generate a sample containing at least one target substance from a first liquid volume of a sample liquid containing the at least one target substance by concentrating the at least one target substance in the first liquid volume. The sample is generated by: - adding a superabsorbent to the first liquid volume or adding the first liquid volume to the superabsorbent, - Incubating the first mixture formed from the superabsorbent and the liquid volume, and - Taking a sample of the liquid portion of the first mixture after incubation.

[0011] Another kit according to the invention is designed to generate a sample containing at least one target substance from a first liquid volume of a sample liquid containing at least one target substance. The sample is generated by: - adding a superabsorbent to the first liquid volume or adding the first liquid volume to the superabsorbent, - incubating the first mixture formed from the superabsorbent and the first liquid volume, in particular until the liquid portion of the first mixture has essentially disappeared, - then adding a second liquid volume of an aqueous solution to the remaining superabsorbent or adding the remaining superabsorbent to the second liquid volume and thus producing a second mixture of the superabsorbent and the second liquid volume, and - Taking a sample of the liquid portion of the second mixture.

[0012] The second mixture formed from the superabsorbent and the second liquid volume can be incubated before taking the sample of the liquid portion of the second mixture, for example to adjust a specific concentration range of the target substance or a specific volume of the liquid portion.

[0013] The aqueous liquid added after incubation of the first mixture may, for example, comprise a lysis buffer.

[0014] The incubation of the first mixture formed from the superabsorbent and the first liquid volume can be continued until the liquid portion of the first mixture is significantly reduced. The liquid portion can also disappear completely or at least be reduced to such an extent that no more liquid can be removed from the mixture using a pipette or swab.

[0015] Using both kits according to the invention, a sample containing at least one target substance is generated, which can subsequently be analyzed and / or further used as a crude product or product in a process or method. Here and in the following, the term sample therefore refers to a volume of substance, for example a liquid volume, which contains the target substance in a concentration that can be influenced or adjusted during the generation of the sample according to the invention. The sample can therefore not only be available for subsequent analysis, but can also be used as a crude product or product for further research purposes or for, for example, industrial product production or for therapeutic purposes. Such samples serving as a crude product or product can, for example, comprise concentrated viruses, nucleic acids, antibodies or proteins.

[0016] Superabsorbents (also known as superabsorbent polymers, SAP) are plastics capable of absorbing many times their own weight in polar liquids. Polar solvents such as water or aqueous solutions are suitable liquids for absorption by the superabsorbent. Upon absorbing the liquid, the superabsorbent swells and forms a hydrogel. Hydrogels can be formed from any crosslinked polar polymer, e.g., polyacrylamide, polyvinylpyrrolidone, amylopectin, gelatin, or cellulose. For the present invention, however, plastics, in particular those mentioned here and below, are preferred over biological polymers.

[0017] Suitable for the invention is, for example, a copolymer of acrylic acid (propenoic acid, H 2 C=CH-COOH) or sodium acrylate (sodium salt of acrylic acid, H 2C=CH-COONa) on the one hand and acrylamide on the other, whereby the ratio of the two monomers to each other can vary. Other polyacrylates or other polymers or copolymers based on acrylic acid or acrylate as a monomer are also possible. In addition, a so-called core crosslinker (CXL) can be added to the monomer solution during copolymer production. This core crosslinker (CXL) connects the long-chain polymer molecules formed to one another at certain points using chemical bridges (crosslinks). These bridges make the polymer insoluble in water. This so-called base polymer is optionally subjected to a so-called surface crosslinking (SXL). In this process, another chemical is applied 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 so that it stays together even under external stress (movement, pressure).

[0018] Superabsorbent granules are commonly used in baby diapers, menstrual hygiene products, incontinence care, and bandages. It is also known to be used in cable sheathing for deep-sea cables.

[0019] Superabsorbents are also used as gel-forming extinguishing agents in firefighting, as mechanical stabilizers for cut flowers in vases, or as additives to potting soil for long-term water retention. Acrylic acid neutralized with potassium hydroxide is used for this purpose due to its greater environmental compatibility. In the form of spherical particles, superabsorbents are 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 small beads of variable sizes from submillimeters to centimeters.As will be described in more detail below, it has surprisingly been found that during the incubation of a mixture of such a superabsorbent and a sample liquid containing at least one, in particular biological, target substance, although a high proportion of the liquid is absorbed by the superabsorbent, the target substance remains in the liquid portion not absorbed by the superabsorbent or, if the liquid portion disappears essentially completely, remains on the surface of the superabsorbent.Since the target substances, in particular biological target substances, are not absorbed by the superabsorbent, the concentration of the at least one target substance in a liquid portion of the first mixture remaining after incubation or the concentration of the target substance in a solution formed by adding an aqueous solution to the superabsorbent remaining after incubation of the first mixture is essentially proportional to the concentration of the target substance in the original sample liquid. This allows a quantitative determination of the concentration of the target substance in the sample liquid based on the generated sample. This effect can also be used to specifically adjust a desired concentration of the target substance in a sample that serves as a raw product or product for further research purposes, therapeutic purposes, or product manufacturing.

[0020] The kits may comprise at least one container containing superabsorbent into which an initial volume of liquid can be added for concentration.

[0021] In the kits according to the invention, the first liquid volume can contain a polar liquid, in particular as the main component. In an advantageous embodiment, the first liquid volume can contain a polar solvent, in particular as the main component. For example, the first liquid volume can consist of a polar solvent to a mass fraction of at least 50%. The polar liquid or polar solvent can be water, for example.

[0022] The target substance can be a biomolecule. It can be selected from the following substances: 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.

[0023] As mentioned, in an advantageous embodiment the superabsorbent can be a plastic or comprise a plastic which absorbs a portion of the liquid volume, e.g. a polar solvent such as water contained in the liquid volume, to form a gel or hydrogel. The plastic is advantageously selected such that it absorbs essentially no biomolecules or the substances specified above such as 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. The superabsorbent should therefore not absorb the target substance, e.g. biomolecules, or should absorb it only to a negligible extent for the purpose of enriching the target substance in the sample to be produced. This is the case, for example, with the aforementioned superabsorbents made from the aforementioned polymer orThis is the case with copolymer materials, e.g. commercially available water pearls, waterbeads, etc.

[0024] The superabsorbent can be used in the form of particles, e.g., as a powder, as granules, or in the form of geometric bodies, especially spheres (spherical particles). It can thus be added to the liquid volume in the form of such particles, or the liquid volume can be added to the superabsorbent in this form. The particles or spheres can have a diameter between 100 and 5000 µm.

[0025] Advantageously, the superabsorbent is in the form of commercially available superabsorbent beads, for example superabsorbent beads available under the names “Aquabeads”, “Water Beads”, “Water Pearls”, “Aqua Pearls”, “Hydro Beads”, “Gel Beads”.

[0026] In an advantageous embodiment, the volume of the liquid portion of the above-mentioned first and / or second mixture remaining after the incubation step, and thus the concentration of the target substance in the remaining liquid portion, can be controlled by the duration of the incubation, by the size and number of the superabsorbent particles or superabsorbent spheres made of the superabsorbent added to the liquid volume, and / or by the temperature prevailing during the incubation.

[0027] The sample obtained using the kits can be manually or automatically transferred to a laboratory device for further processing or analysis. Further analysis can be performed using molecular genetic analysis techniques, immunological technologies, and spectroscopic technologies, e.g., molecular spectroscopy or mass spectroscopy; sensor-based methods, e.g., optical or electrochemical sensors; cultivation, sequencing, or flow cytometry.

[0028] For the qualitative or quantitative determination of at least one target substance in a sample fluid, at least one of the following methods can be used: nucleic acid-based detection methods, sequencing, immunological detection methods, microbiological analyses, microscopic methods, mass spectrometry, sensor-based detection and flow cytometric techniques.

[0029] In addition to the superabsorbent, which is available in a dosage form suitable for the procedure, the kit may contain other chemicals, such as buffer solutions or lysis buffers.

[0030] Nucleic acid-based detection methods can include PCR, real-time PCR, digital PCR-based methods, or sequencing. Immunological detection methods can be immunological assays such as ELISA or lateral flow tests. Microbiological analyses can involve the cultivation of living cells in the sample. Sensor-based detection methods can include detection methods using optical, spectroscopic, or electrochemical sensors.

[0031] In one possible embodiment, the generated sample or the further processed sample can be placed into a cartridge, in particular a microfluidic cartridge, of an automated analysis device for automated detection of the target substance using molecular genetic techniques such as PCR or real-time PCR. This can be done manually or automatically.

[0032] The invention is explained in more detail below with reference to the figures and some exemplary embodiments. These examples do not represent a limitation of the inventive compositions.

[0033] They show: Fig. 1 a schematic representation of a liquid before (a) and after adding a superabsorbent in the form of spheres and incubating (b); Fig. 2 Amplification curves of various samples from surface water without concentration and after concentration using the kit according to the invention; Fig.3 Amplification curves of various samples from water containing Salmonella without concentration, after concentration by a filtration process and after concentration by means of the kit according to the invention; Fig. 4 Amplification curves of various samples from water containing MS2 phage RNA without concentration, after concentration by a filtration method and after concentration by the kit according to the invention; Fig. 5 a gel electrophoretic representation of genomic DNA from a water sample without concentration and after concentration using the kit according to the invention; Fig. 6 a gel electrophoretic representation of the DNA of eukaryotic cells from a water sample without concentration and after concentration using the kit according to the invention; and Fig.7 Amplification curves of different samples of water containing DNA at very low concentrations, without concentration and after concentration by means of the kit according to the invention using superabsorbent beads of different sizes.

[0034] 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 many times their original volume. The volume of the liquid portion of the mixture consisting 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.

[0035] A 50 ml reference sample was taken directly from the liquid, i.e., the aforementioned surface water, without prior concentration using the described kit. Both samples were centrifuged at 5000 × g 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.

[0036] 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 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 concentrated, and the biomolecules were concentrated in the remaining liquid. Even more surprising was that the described kit can also be used to concentrate proteins and free genomic DNA, which are present in low concentrations in an aqueous solution.

[0037] The use of superabsorbents to concentrate a target substance, especially biomolecules, in a polar liquid as a solvent, for example water, is very simple and universally applicable. A suitable method is based on Fig. 1 briefly described as follows: 1. Addition of a superabsorbent 2 to a volume of a, in particular aqueous, liquid 1, or alternatively: addition of the liquid to a superabsorbent 2 provided; 2. Incubation of the mixture of the liquid 1 and the superabsorbent 2 to reduce the volume of the liquid portion 3 of the mixture; and then 3. Transfer at least a portion of the liquid portion 3 of the mixture into a new container as a sample for further processing. Further processing can be, for example, nucleic acid extraction, measurement, or direct analysis using a variety of technologies, such as NGS applications, immunological technologies, spectroscopic technologies, molecular spectroscopic or mass spectrometric technologies, etc.

[0038] Alternatively, the transferred part of the liquid fraction can also be used as a product for further research purposes, for therapeutic purposes or as a raw product in a production process.

[0039] The degree of concentration and the speed of this process can be controlled very precisely by the type of superabsorbent used, by its amount used, or by the incubation time and / or the incubation temperature.

[0040] The kit thus provides a simple solution to the problem of processing low-concentration samples for further analysis of the target substances of interest, particularly biomolecules, or for their detection. The kit does not require expensive equipment such as ultracentrifuges, expensive ultrafiltration membranes, complex procedures such as PEG precipitation, or general precipitation reactions for concentrating nucleic acids, etc. Furthermore, the kit is universally applicable with regard to the target substances, particularly biomolecules. A further advantage is that the superabsorbents are non-toxic and harmless, and often even biodegradable. The kit according to the invention can therefore greatly simplify the investigation of low-concentration biomolecules.

[0041] Further experiments on concentrating genomic DNA contained in a sample liquid revealed yet another effect. In these experiments, an aqueous DNA solution with a volume of 500 µl was incubated with the addition of a sphere made of superabsorbent material (commercially available water beads; diameter approx. 1 mm). With increasing incubation time, the sample volume decreased and the DNA concentration increased. The process was concluded when the volume of the liquid was de facto zero. This result implied the idea of ​​DNA loss. All the more surprising was the observation that in a sample generated by adding 250 µl of water to the sphere made of superabsorbent material and briefly shaking the container, DNA was measurable at approximately twice the concentration compared to the initial sample of 500 µl.This demonstrated that the DNA was not absorbed by the superabsorbent material, but rather had accumulated on the outer surface of the superabsorbent sphere and could be recovered after adding a liquid medium. This makes it possible not only to concentrate biomolecules in a sample liquid using the kit according to the invention, but also to specifically adjust the biomolecules, in this case DNA, to a desired concentration by adding an aqueous solution after the liquid has been completely absorbed by the absorber used.

[0042] Based on this observation, further possibilities for using superabsorbents to concentrate biomolecules have emerged. The following method can be used to generate a sample of a sample liquid that can be used for qualitative or quantitative analysis and contains biomolecules such as eukaryotic cells, prokaryotic cells, viruses, phages, or subcellular compartments as the target substance to be determined qualitatively or quantitatively: First, an initial volume of the sample liquid can be treated with a superabsorbent such that the entire liquid is absorbed by the superabsorbent. The preferred superabsorbent is the so-called water beads described above. After incubation and complete absorption of the liquid, a lysis buffer is added to the superabsorbent, and the resulting mixture is incubated. The type of lysis buffer and / or the incubation time can be freely selected.The lysis buffer is then separated from the superabsorbent. The resulting liquid sample contains the target substance. The biomolecules contained in the sample as the target substance can be further lysed if necessary. After lysis, the sample can be used for nucleic acid extraction. Provided that lysis has already been successfully completed, the lysate can be used for nucleic acid extraction without further incubation.

[0043] In the following, some embodiments of the invention are described in more detail. Example 1: Concentration of a wastewater sample with a volume of 1 liter

[0044] Wastewater from a fire pond was used as the sample fluid. Two volumes of 1 liter of the fluid were transferred into a sample bottle.

[0045] For the concentration test, 50 g of commercially available superabsorbent beads known as "Water Beads," with a mass of approximately 0.006 g per bead and a diameter of approximately 1 mm, were added to one of the sample bottles. After incubation, the remaining liquid portion of approximately 650 ml was transferred to a new container. This reduced the original volume of sample liquid from 1000 ml to 650 ml. To demonstrate that the concentration of bacteria in the concentrated sample liquid could be increased, two samples (samples 1 and 2) with a volume of 10 ml of the concentrated sample liquid and, for comparison, two samples of the unconcentrated sample liquid contained in the second sample bottle (samples 3 and 4) were centrifuged in a 15 ml reaction tube at 5000 rpm for 10 min, and the supernatant was removed. The resulting sediment pellet was subsequently used for DNA extraction.DNA extraction was performed using a commercial kit (innuPREP Stool DNA Kit; IST Innuscreen GmbH). The extracted DNA was used to determine the total bacterial count using real-time PCR. A commercial kit (innuDETECT Bacteria Quantification Assay; IST Innuscreen GmbH) was used as the detection system.

[0046] The standard DNA from the assay was used to determine the number of bacterial copies in the sample.

[0047] In Fig. Figure 2 shows the amplification curves of the samples. Curves A (solid line) are the amplification curves of the three standards. Curves B (long and short dashes) are the amplification curves of samples 1 and 2 taken from the concentrated sample fluid, and curves C (equal-length dashes) are the amplification curves of samples 3 and 4 taken from the untreated sample fluid. Table 1 shows the Ct values ​​and copy numbers for the individual standards and samples.

[0048] The results of the real-time PCR demonstrate that by using the kit according to the invention, an approximately 3-fold concentration of the total number of germs in the sample can be detected. Table 1: sample Ct value Number of copies 1 (after constriction) 21,2 4,3×10 5 2 (after constriction) 20,7 6,1×10 5 3 (not restricted) 23,3 1,1×10 5 4 (not restricted) 22,8 1,5×10 5 Standard 1 18,9 2×10 6 Standard 2 22,2 2×10 5 Standard 3 25,6 2×10 4 Example 2: Detection of Salmonella in water samples

[0049] Tap water with added salmonella was used as the sample liquid, with 1 × 10 6 Salmonella was added as a spike.

[0050] From such an unconcentrated sample volume, two samples (Sample 1 and 2) of 200 µl each were taken as reference samples. Two further sample volumes of 10 ml were concentrated using the kit according to the invention. For the concentration, superabsorbent beads commercially available under the name "Water Beads" were used. Each bead has a mass of approximately 0.006 g and a diameter of approximately 1 mm. Forty "Water Beads" were added to each of the two sample volumes. By incubating the resulting mixture, its liquid portion was concentrated to a volume of approximately 500 µl. Of this liquid portion, 2 × 200 µl each were used for the subsequent DNA extraction (samples 3 to 6).

[0051] In parallel, another 10 ml sample volume was processed using a standard procedure using a filtration membrane. The sample volume was filtered through a filter membrane (0.45 µm MCE membrane; Millipore) using a vacuum pump. The filter was cut, mixed with 600 µl of 1 × PBS solution, and homogenized in a lysis tube using a homogenizer (SpeedMill, Analytik Jena GmbH). From the approximately 500 µl liquid obtained after homogenization, two samples with a volume of 200 µl each were also used for DNA extraction (samples 7 and 8).

[0052] DNA extraction was performed using an automated procedure on the KingFisher Flex (Thermo Fisher) and a commercially available kit (deltaPREP AniPath DNA / RNA Kit KFFLX; IST Innuscreen GmbH). The extracted DNA was used for Salmonella detection using real-time PCR. A commercial kit (innuDETECT Salmonella enterica Assay; IST Innuscreen GmbH) was used as the detection system.

[0053] In Fig. Figure 3 shows the amplification curves of the samples. Curves A (long and short dashes) show the amplification curves of samples 1 and 2 of the unconcentrated sample fluid. Curves B (equal-length dashes) are the amplification curves of samples 3 to 6 of the concentrated sample fluid. Curves C (solid line) are the amplification curves of samples 7 and 8, which were obtained by filter-based enrichment.

[0054] Table 2 shows the Ct values ​​for the individual samples. Table 2: sample Ct value 1 (without constriction) 33,3 2 (without constriction) 33,1 3 (after constriction) 28,4 4 (after constriction) 28,3 5 (after constriction) 28,2 6 (after constriction) 28,2 7 (Filter-based) 29,9 8 (Filter-based) 29,4

[0055] The differences in the Ct values ​​indicate a calculated 32-fold concentration of the initial sample in terms of Salmonella count. The enrichment of the bacteria on a filter shows a lower efficiency compared to the sample obtained using the kit according to the invention. Example 3: Detection of MS2 phage RNA in water samples

[0056] Tap water with added MS2 bacteriophages was used as sample liquid, with 5 µl of an MS2 bacteriophage solution (Leibniz Institute DSMZ: DSM 13767) being added as a spike to each volume of 10 ml of tap water.

[0057] From this sample volume, two samples (Samples 1 and 2) of 200 µl each were taken as reference samples. Two further sample volumes of 10 ml each were concentrated using the kit according to the invention. Superabsorbent beads commercially available under the name "Water Beads" were used for the concentration. Each bead weighs approximately 0.006 g and has a diameter of approximately 1 mm. 40 "Water Beads" were added to each of the two sample volumes. By incubating the resulting mixture, its liquid portion was concentrated to a volume of approximately 500 µl. Of this liquid portion, two 200 µl portions were used for the subsequent phage RNA extraction (Samples 3 to 6).

[0058] In parallel, another 10 ml sample volume was processed using a standard procedure using a filtration membrane. The sample volume was filtered through a filter membrane (0.45 µm MCE membrane; Millipore) using a vacuum pump. The filter was cut, mixed with 600 µl of 1 × PBS solution, and homogenized in a lysis tube using a homogenizer (SpeedMill, Analytik Jena GmbH). Of the approximately 500 µl sample volume obtained after homogenization, two samples, each with a volume of 200 µl, were used for phage RNA extraction (samples 7 and 8).

[0059] Phage RNA extraction was performed using an automated procedure on the KingFisher Flex (Thermo Fisher) and a commercially available kit (deltaprep AniPath DNA / RNA Kit KFFLX; IST Innuscreen GmbH). The extracted phage RNA was used to detect MS2 phage RNA using real-time PCR. A commercial kit (innuDETECT Internal Control DNA / RNA Assay; IST Innuscreen GmbH) was used as the detection system. A commercial one-step RT master mix (innuDRY qRT-PCR MasterMix Probe; IST Innuscreen GmbH) was used for reverse transcription and amplification of the MS2 phage RNA.

[0060] In Fig.Figure 4 shows the amplification curves of the samples. Curves A (long and short dashes) indicate the amplification curves of samples 1 and 2 of the unconcentrated sample fluid. Curves B (equal-length dashes) are the amplification curves of samples 3 to 6 of the concentrated sample fluid. Curves C (solid line) are the amplification curves of samples 7 and 8, which were obtained by filter-based enrichment.

[0061] Table 3 shows the Ct values ​​for the individual samples: Table 3: sample Ct value 1 (without constriction) 30,8 2 (without constriction) 31,3 3 (after constriction) 24,6 4 (after constriction) 25,1 5 (after constriction) 24,6 6 (after constriction) 24,5 7 (Filter-based) 35,7 8 (Filter-based) 35,4

[0062] The differences in Ct values ​​indicate a 50-fold concentration of the original sample. Filter-based enrichment appears to be very poorly suited for phage enrichment. Example 4: Concentration of genomic DNA in a water sample and spectrophotometric measurement

[0063] To demonstrate that the kit according to the invention is also suitable for concentrating genomic DNA in a sample liquid, genomic DNA was isolated from a blood sample. The DNA was dissolved in water, and the DNA concentration was adjusted to 10 ng / µl. The initial volume of the sample liquid thus prepared was 2 ml. Concentration was carried out by adding 10 commercially available "water beads" (mass per piece approx. 0.006 g; diameter: approx. 1 mm). After a short incubation period, the volume of the sample liquid was concentrated to a residual volume of 500 µl, and a first spectrophotometric measurement was performed to determine the DNA concentration in the liquid portion of the mixture. After extending the incubation time, the liquid portion of the mixture was further concentrated to 250 µl, and a second spectrophotometric measurement was performed to determine the DNA concentration in the liquid portion of the mixture.The results are summarized in Table 4. Table 4: sample DNA concentration Sample liquid before concentration 10 ng / µl sample liquid concentrated to 500 µl 37 ng / µl Sample liquid concentrated to 250 µl 70 ng / µl

[0064] The data show that after concentration, the measured values ​​correspond to the theoretical values ​​with only a small deviation. Example 5: Concentration of genomic DNA from a 2 ml sample and detection of the increase in concentration on an agarose gel

[0065] To demonstrate that the kit according to the invention is also suitable for concentrating genomic DNA from a sample fluid, genomic DNA was isolated from a blood sample. The DNA was dissolved in water, and the DNA concentration was adjusted to 10 ng / µl. The initial volume of the sample fluid thus prepared was 2 ml. From this initial volume, a first sample was taken as a reference sample for gel electrophoresis. The initial volume was reduced by adding 10 commercially available "water beads" (mass approx. 0.006 g; diameter: approx. 1 mm). After a short incubation period, the liquid portion of the mixture thus produced was concentrated to a residual volume of 250 µl. A second sample taken from this residual volume was subsequently visualized on an agarose gel in comparison to the initial sample.

[0066] Fig.Figure 5 shows the gel electrophoresis of the DNA. The first lane (1) contains the DNA ladder, the second lane (2) the reference sample, and the third lane (3) the second sample from the concentrated residual volume. The gel image clearly shows the greatly increased amount of DNA after concentration compared to the unconcentrated sample fluid. Example 6: Enrichment of eukaryotic cells in a sample fluid and subsequent extraction of DNA from these cells

[0067] To demonstrate that the kit according to the invention is also suitable for concentrating eukaryotic cells present in a sample fluid, nucleated cells were isolated from a blood sample. The cells were then completely resuspended in an initial volume of 2 ml of water. Before concentrating the sample fluid, 200 µl of the initial cell suspension was taken as a reference sample and used for DNA extraction.

[0068] The sample fluid was concentrated by adding eight superabsorbent beads, commercially available under the name "Water Beads," which have a mass of approximately 0.009 g and a diameter of approximately 2 mm. After a short incubation period, the initial volume of the sample fluid was concentrated to a residual volume of approximately 400 µl. Of this approximately 400 µl, 200 µl were taken as a sample and used for DNA extraction. DNA extraction was performed using a commercial kit (innuPREP DNA Mini Kit 2.0; IST Innuscreen GmbH). DNA was measured spectrophotometrically and analyzed on an agarose gel.

[0069] The results of the spectrophotometric measurements and the determined amount of DNA in the respective samples are summarized in Table 5. Table 5: sample DNA quantity Ratio A 260 :A 280 Ratio A 260 :A 230 Extraction from 200 µl of the initial volume (reference sample) 2,1 µg 1,8 2,2 Extraction from 200 µl of the residual volume concentrated to approximately 400 µl 9,9 µg 1,9 2,3

[0070] Fig. Figure 6 shows the gel electrophoretic representation of the DNA, with the DNA ladder in the first lane (1), the control sample in the second lane (2) and the second sample from the concentrated residual volume in the third lane (3).

[0071] The data demonstrate that eukaryotic cells can be enriched from a sample using the kit of the invention. The DNA is of high quality. Example 7: Concentration of a protein solution

[0072] To demonstrate that the kit according to the invention is also suitable for concentrating proteins in a sample liquid, an aqueous albumin solution was prepared. The protein concentration in the untreated starting solution was determined spectrophotometrically at 280 nm to be 11.8 mg / ml. Different concentrations were carried out. For this purpose, an initial volume of 100 µl of the protein solution was transferred to a reaction vessel and the solution was incubated with a sphere of commercially available "water beads" (mass approx. 0.006 g; diameter: approx. 1 mm) for different periods of time. This resulted in the concentration of the starting solution from 100 µl to approx. 60 µl, approx. 40 µl and approx. 20 µl. The residual volumes thus concentrated were subsequently measured as individual samples at 280 nm and the protein concentration was determined. The results are summarized in Table 6. Table 6: sample Protein concentration determined from photometric measurement at 280 nm in mg / ml Initial solution 11,8 Sample concentrated to approx. 60 µl 18,2 Sample concentrated to approx. 40 µl 24,1 Sample concentrated to approx. 20 µl 46,3

[0073] It turns out that proteins can also be concentrated using the kit and that the concentrations increase continuously depending on the concentration. Example 8: Increase in sensitivity of real-time PCR after concentration of a sample with very low concentration of human DNA

[0074] To demonstrate that the kit according to the invention is also suitable for concentrating solutions with very low DNA concentrations, a sample liquid containing human genomic DNA at a very low concentration was prepared. The DNA concentration of the sample was 8 ng / µl, which corresponds approximately to the amount of genomic DNA in a diploid eukaryotic cell. For concentration, 100 µl of the sample liquid was used as the starting volume. One commercially available "Water Beads" (mass approx. 0.006 g; diameter: approx. 1 mm) was added to a first starting volume of the sample liquid, and one commercially available "Water Beads" (mass approx. 0.009 g; diameter: approx. 2 mm) was added to a second starting volume of the sample liquid. After a brief incubation, the liquid portions of the two mixtures were concentrated to approximately 10 µl.A reference sample taken from the non-concentrated sample fluid (sample 1) and samples taken from the concentrated liquid portions of the two mixtures (sample 2-5) were used in real-time PCR to detect a human-specific target sequence (single copy gene estrogen receptor 1, in-house method).

[0075] The amplification curves of the individual samples are shown in Fig. 7. Curves A (solid line) color are the amplification curves of the control sample (duplicate determination), curves B (long and short dashes) are the amplification curves of samples 2 to 5.

[0076] Table 7 shows the Ct values ​​for the individual samples. Table 7: sample Ct value 1 (before constriction) No Ct 1 (before constriction) No Ct 2 (after constriction Ø 1mm “Water Beads”) 38,2 3 (after constriction Ø 1mm “Water Beads”) 37,4 4 (after constriction Ø 2mm “Water Beads”) 38,4 5 (after constriction Ø 2mm “Water Beads”) 38,5

[0077] Taking a sample from the starting solution did not yield an amplification result because the DNA concentration was too low. The DNA from the concentrated samples was able to detect the single copy gene, demonstrating the success of the kit according to the invention. Example 9: Generation of a sample by incubating a DNA solution with a superabsorbent until complete liquid absorption and subsequent adjustment of the sample concentration by adding an aqueous solution

[0078] A DNA solution with a concentration of 100 ng / µl (lambda DNA) was prepared as the sample fluid. Two commercially available water beads (0.006 g; diameter approximately 1 mm) were added to 500 µl of this DNA solution, and the resulting first mixture was incubated until the liquid portion of the mixture had completely disappeared. 250 µl of a buffer (10 mM Tris HCl, pH 8.5) was then added to the remaining water beads. The resulting second mixture was mixed using a vortex mixer, and the liquid portion of the second mixture was then separated from the water beads as the sample to be analyzed and analyzed spectrophotometrically.

[0079] Table 8 shows the original DNA concentration in the original DNA solution used as sample liquid and the spectrophotometrically determined concentration in the sample. Table 8: sample DNA concentration Sample liquid 500 µl 100 ng / µl Sample after complete liquid reduction and subsequent addition of 250 µl Tris buffer 181 ng / µl

[0080] The data show that the concentration determined in the sample after concentration and resuspension corresponds to the theoretical values ​​with only slight deviation. Example 10: Concentration of a sample liquid for the detection of a protein (CRP) using an immunological detection method

[0081] A dilution of C-reactive protein (CRP) in a PBS buffer solution was used as the sample fluid. Immunological detection of C-reactive protein in various samples generated from the sample fluid was performed using a CRP ELISA kit from Bio-Techne GmbH (h C-Reactive Protein DuoSet, DY1707). The kit's positive standard control (human CRP) served as the starting sample. Measurements were performed in an ELISA reader (Thermofisher) at 405 nm. Three solutions (samples 1, 2, and 3) with different CRP concentrations were generated from the starting sample. An initial portion of approximately 80 µl was taken from each of these samples, diluted with 120 µl of the kit's dilution buffer, and applied to the ELISA plate. A further portion of 500 µl each was taken from samples 1, 2 and 3, mixed with 6 “Water Beads” (0.006 g, diameter 1 mm) each and incubated until the resulting samples 1A, 2A and 3A had a volume of approx.70-80 µl were concentrated. These concentrated samples 1A, 2A, and 3A were also diluted with 120 µl of the kit's Dilution Buffer and applied to the ELISA plate. The solutions from samples 1, 2, 3, 1A, 2A, and 3A applied to the ELISA plate were measured semiquantitatively alongside a standard series [S1-S4, 200 µl each] according to the manufacturer's instructions. The results are summarized in Table 9. Table 9: Sample No. / St No. CRP concentration (pg / ml) (theoretically calculated for samples 1A-3A) CRP amount (pg) (Theoretically calculated for samples 1A-3A) Measurement at 405 nm CRP concentration calculated from measurement 1 12000 960 0,710 3700 2 1200 96 0,185 367 3 120 9,6 0,079 nD 1A 75000 6240 1,006 7800 2A 7500 624 0,482 2000 3A 750 62,4 0,088 120 S1 2000 40 0,484 2000 S2 1000 20 0,322 1000 S3 500 10 0,215 500 S4 250 5 0,152 250

[0082] Due to the insufficient binding capacity, range, and linearity of the ELISA method, the theoretically expected protein amounts could not be measured precisely. The theoretically calculated enrichment was a factor of 6.5. The enrichment rate measured by ELISA ranges between 2.1 and 5.4.

[0083] The concentration of the non-measurable sample 3 could be determined in sample 3A, which was generated from sample 3 by enrichment. This demonstrates that using the kit according to the invention, it is possible to concentrate a specific target protein in a sample fluid and detect it by ELISA. Thus, a sensitivity advantage can be achieved through concentration. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This 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

[0000] US 2015 / 0224502 ​​A1

[0006] Cited non-patent literature

[0000] Yamamoto et al., Virology 40 (1970) 734

[0005] Morandi et al., J. Clin. Microbiol. 36 (1998) 1543-1538

[0005]

Claims

[1] Superabsorbent (2) which comprises a plastic which absorbs water to form a hydrogel and which absorbs essentially no biomolecules, in particular essentially no eukaryotic cells, components of eukaryotic cells, prokaryotic cells, components of prokaryotic cells, subcellular vesicles, bacteriophages, viruses or virus components, toxins, antibodies, nucleic acids or proteins. [2] Kit comprising a superabsorbent (2) according to claim 1, wherein the superabsorbent (2) is designed to produce a sample containing at least one target substance from a first liquid volume (1) of a sample liquid containing the at least one target substance by concentrating the target substance in the first liquid volume (1), wherein the superabsorbent is added to the first liquid volume or wherein the first liquid volume is added to the superabsorbent, wherein the first mixture formed from the superabsorbent (2) and the first liquid volume (1) is incubated, and wherein a sample of the liquid portion (3) of the first mixture present after the incubation is taken. [3] A kit comprising a superabsorbent (2) according to claim 1, wherein the superabsorbent (2) is designed to produce a sample containing at least one target substance from a first liquid volume (1) of a sample liquid (1) containing the at least one target substance, wherein the superabsorbent is added to the first liquid volume or wherein the first liquid volume is added to the superabsorbent, wherein the first mixture formed from the superabsorbent (2) and the first liquid volume (1) is incubated, wherein thereafter a second liquid volume of an aqueous solution is added to the remaining superabsorbent or the remaining superabsorbent is added to the second liquid volume and a second mixture of the superabsorbent and the second liquid volume is produced, and wherein a sample of the liquid portion of the second mixture is taken. [4] Kit according to claim 2 or 3, wherein the second mixture formed from the superabsorbent and the second liquid volume is incubated before taking the sample of the liquid portion of the second mixture. [5] Kit according to any one of claims 2 to 4, wherein the aqueous solution comprises a lysis buffer. [6] Kit according to any one of claims 2 to 5, wherein the incubation of the first mixture is carried out until the liquid portion of the first mixture has substantially completely disappeared. [7] Kit according to one of claims 2 to 6, wherein the first liquid volume (1) contains a polar liquid, in particular as the main component. [8] Kit according to claim 7, wherein the polar liquid is water. [9] Kit according to any one of claims 2 to 8, wherein the target substance is a biomolecule. [10] Kit according to any one of claims 2 to 9, wherein the target substance is 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. [11] Kit according to any one of claims 2 to 10, wherein the volume of the liquid portion of the first or second mixture remaining after incubation is controlled by the duration of the incubation and / or by the type and / or amount of superabsorbent and / or by the temperature of the mixture prevailing during the incubation. [12] Kit according to one of claims 2 to 11, wherein the superabsorbent (2) is used in the form of particles, e.g. as a powder, as granules or in the form of geometric bodies, in particular spheres, and wherein the volume of the liquid portion (3) remaining after incubation is controlled by the size and / or number of the particles. [13] Kit according to claim 12, wherein the superabsorbent is used in the form of commercially available water beads, hydrospheres, aquabeads, waterbeads, or gel beads. [14] Kit according to any one of claims 2 to 13, wherein the sample produced is used as a raw product or product in an experimental procedure or in a production process. [15] Kit according to one of claims 2 to 14, wherein a qualitative or quantitative detection of the at least one target substance is carried out on the basis of the sample by means of at least one of the following methods: nucleic acid-based detection methods, in particular PCR, real-time PCR or digital PCR-based methods, sequencing, immunological detection methods, in particular ELISA, lateral flow tests, microbiological analyses, microscopic methods, mass spectrometry, detection methods using optical, spectroscopic or electrochemical sensors, and flow cytometry. [16] Kit according to one of claims 2 to 15, further comprising further chemicals, such as buffer solutions or lysis buffers, and / or at least one container into which the superabsorbent (2) is placed, which container is designed for adding the first liquid volume for concentration.

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