DE102015210395A1 - Filter device and a method for the selective separation of an analyte from a liquid - Google Patents

Abstract

The present invention relates to a filter device and a method for the selective separation of an analyte from a liquid, wherein the following steps according to the invention comprises: a) contacting a filter device comprising a separation material, which is designed as a nanocomposite polymer network for ion exchange, with a liquid containing an analyte and at least one further component, wherein the analyte absorbs to the nanocomposite polymer network, b) separation of the analyte-free liquid containing at least the one further component from the separating material, wherein the separating material under standard conditions has a wetting factor φ greater than 0.95.

Description

The present invention relates to a filter device and a method for the selective separation of an analyte from a liquid.

At present, only polymer-based structures are known from the prior art, which are formed as charge-based barrier layers with a positive or negative excess charge. An absorption of substances is not provided.

For example, are off EP 2 315 815 A1 Polyelectrolyte complexes with fat barrier properties known that carry an anionic charge at pH <6.0.

Already Kaiser et al. (Biochim Biophys Acta 1971, 232 (2), 388-402) described the use of clay minerals for the selective removal of ribozymes from cell lysates.

EP 0 937 735 A1 discloses the selective removal of pathogenic virus proteins (NCTA) from a protein solution by adsorption on kieselguhr, diatomaceous earth, clay minerals, metal hydroxides or oxyhydrate.

Out US 2014/0158619 A1 a filter device for the separation of unwanted proteins from solutions is known in which clay minerals of the montmorillonite type are immobilized on a membrane or arranged as a loose bed between two membranes or a filter housing. The separation is irreversible.

Working with clay minerals in all these processes is particularly disadvantageous because it is free-flowing in the dry state as a particulate bulk material or diffusely dispersed in the dispersed state. In order to prevent or at least minimize the discharge of particularly small clay mineral particles, the particle bedding or particle dispersion must always be stored in a stationary manner by the use of close-meshed grid elements or by means of membranes.

In addition, disadvantageous the known particle beds in the contacting of the clay mineral particles with a liquid gas inclusions or tend to stick / lumping (see. 4 , left side), so that under standard conditions (T = 25 ° C, p = 101.3 Pa) the entire surface of the clay mineral particles is prevented from being used for the adsorption of matter. This circumstance is taken into account by the wetting factor φ. The wetting factor φ (as a measure of the actual adsorbent wetted surface of the clay mineral particles) is calculated as follows: φ = actually wetted surface / theoretically wettable surface

The particle beds known from the prior art have only a wetting factor φ less than 1 under standard conditions. In order to achieve a homogeneous distribution of the liquid between the clay mineral particles and bring the wetting factor φ close to 1, the sample must be subjected to negative pressure or centrifuged.

It is therefore an object of the present invention to provide a filter device based on clay minerals, which allows the selective separation of an analyte from a solution in a simple manner and thereby overcomes the aforementioned disadvantages of the prior art.

The object is achieved by a method according to claim 1 and an apparatus for the selective separation of an analyte from a liquid according to the following claims. Advantageous embodiments are specified in the dependent claims.

• a) Kontaktieren einer Filtervorrichtung umfassend ein Trennmaterial, welches als Nanokompositpolymernetzwerk für den Ionenaustausch ausgebildet ist, mit einer Flüssigkeit enthaltend einen Analyten und zumindest eine weitere Komponente, wobei der Analyt an das Nanokompositpolymernetzwerk absorbiert,
• b) Abtrennung der analytfreien Flüssigkeit, enthaltend zumindest die eine weitere Komponente, von dem Trennmaterial,

wobei das Trennmaterial unter Standardbedingungen einen Benetzungsfaktor φ größer 0,95 aufweist.The method according to the invention for the selective separation of an analyte from a liquid, comprising the steps:

• a) contacting a filter device comprising a release material, which is designed as a nanocomposite polymer network for ion exchange, with a fluid containing an analyte and at least one further component, wherein the analyte absorbs to the nanocomposite polymer network,
• b) separating the analyte-free liquid containing at least the one further component from the separating material,

wherein the release material under standard conditions has a wetting factor φ greater than 0.95.

For the purposes of the present invention, the term "nanocomposite polymer network" refers to a physically crosslinked nanocomposite polymer comprising at least one organic polymer and at least one kind of clay mineral particles selected from the group of swellable phyllosilicates, wherein the clay mineral particles in the nanocomposite polymer network act as crosslinkers (by way of example 4 , right side).

For the purposes of the present invention, "analyte-free" means that at least 70%, preferably at least 85%, particularly preferably at least 95%, of the analyte, based on the concentration of the analyte in the liquid before the time of contacting with the separating material, absorbs from the separating material become.

The clay mineral particles used according to the invention are particularly advantageously incorporated into the nanocomposite polymer network as crosslinker, so that the release material, in contrast to conventional clay mineral-based release materials, does not constitute a free-flowing particle bed. In this way, when used in a filter device, in particular when used in microsystem technology (MST), it is possible to dispense with a membrane or grid element which is upstream and / or downstream of the flow direction.

In a particularly advantageous manner, the separating material can be adapted precisely and finally to the shape of the container (for example cylinder, rectangle, channel).

Particularly advantageously, the nanocomposite polymer networks used according to the invention have a high mechanical stability compared to conventional brittle clay minerals. Thus, the separation material used according to the invention is characterized in particular by an elongation at break (characteristic parameter for the deformability (or ductility) of a material, ie permanent extension of the release material after its fraction ΔL, based on the initial measurement length L ₀ ) of greater than 1500% and a maximum mechanical Tensile stress in the range of 50 to 400 kPa. At the same time, the release material can be compressed over 90%. For determining the elongation at break on the basis of DIN 53504 According to the invention cylindrical sample body with a diameter of 5 mm and the length of 5 cm in the swollen state, ie in equilibrium swelling degree (Q _V between 5 and 100) or in the synthesis state, at room temperature are clamped in a tractor and subjected to a tensile force.

Since the clay mineral particles are completely exfoliated and immobilized in the polymer network and are distributed statistically, they can be completely and completely lapped by the liquid (cf. 4 , right side)

In addition, the swellability of the release material (the ability to uniformly absorb swelling agent) provides the release material with good penetration even under standard conditions such that the clay mineral particles are completely immersed in the liquid and the total specific surface area of the clay mineral particles is usable for the material adsorption , In this way, dead zones (i.e., gas inclusions) in the separating material are particularly advantageously avoided, and a wetting factor φ greater than 0.95 is realized, and thus, in contrast to conventional clay mineral-based separation processes, a significantly larger specific surface area is available. In contrast to conventional clay mineral-particle-based release materials (with identical size, shape and porosity of the particles), it is therefore particularly advantageous for the release materials used according to the invention to require 50% less, preferably 75%, less clay mineral particles per unit volume of the release material.

Particularly preferably, the wetting factor φ of the clay mineral particles in the release material used according to the invention is at least 0.97, very particularly preferably at least 0.98.

By definition, the nanocomposite polymer networks used according to the invention are referred to as solids (also referred to as bulk materials), in contrast to conventional clay mineral-particle-based separation materials in that no dispersion of the clay mineral particles forms by contact with a liquid. Consequently, a discharge of the clay mineral particles from the nanocomposite polymer network is particularly advantageous over the period of the inventive method, so that advantageously the separation effect of the release material remains the same throughout the entire process. Furthermore advantageously, contamination of the sample or of the analyte with clay mineral particles during the process is prevented at the same time.

According to a preferred variant of the present invention are used as liquids hydrophilic liquids, in particular water or hydrophilic organic liquids selected from the group of ethanol, methanol, isopropanol, n-propanol, ammonia, acetone or mixtures thereof.

A disadvantage of the use of conventional clay mineral-based release materials (conventional particle beds) is that the clay mineral particles in organic liquids can not or only with difficulty be dispersed and consequently are only applicable in water.

The use according to the invention of the nanocomposite polymer networks particularly advantageously allows working with mixtures of water and at least one of the abovementioned organic liquids, so that organic materials that are insoluble in water can be dissolved and selectively removed via the process according to the invention. It is known to the person skilled in the art that water and the abovementioned organic liquids are basically miscible with each other in any ratio, so that mixtures of water and organic liquid in a volume ratio of from 1:99 to 99: 1 are preferred.

According to a preferred embodiment of the present invention, the aqueous liquid has a pH in the range from 2 to 12, particularly preferably in the range from 3 to 10.

Optionally, a constant pH can be ensured in the liquid by the addition of buffer components. Preferably, the buffer components are independently selected from the group consisting of Tris, MES, bis-tris, ADA, aces, PIPES, MOPSO, bis-trispropanes, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, trizma, HEPPSO, POPSO , TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, phthalates, acetates, phosphates and citrates, and mixtures thereof. According to a preferred embodiment of the present invention, the concentration of the buffer component is in a range from 0.01 to 0.2 mol / l. The individual abbreviations for the respective buffer components are known to the person skilled in the art.

Preferably, the analyte is selected from the substance group of the biomolecules, particularly preferably a peptide, in particular an oligopeptide (for example messenger substances) or protein (for example antibody or antigen), a constituent of nucleotides (for example adenine, uracil or ribose) or polynucleic acid strand ( For example, DNA, RNA or modified derivatives thereof), wherein the liquid is preferably an aqueous liquid. Most preferably, the biomolecule is a peptide, preferably having a molecular weight in the range of 1100 g / mol to 70000 g / mol or DNA.

According to a preferred embodiment of the present invention, the analyte is a low-molecular-weight organic compound.

For the purposes of the invention, low molecular weight organic compounds are understood as meaning molecular compounds which are composed primarily of the elements carbon, hydrogen, oxygen and nitrogen and preferably have a molecular weight in the range from 100 to 1500 g / mol, in particular from 150 to 1000 g / mol , especially 200 to 750 g / mol. Examples of low molecular weight compounds in the context of the invention are organic dyes, natural products and pharmaceutically active substances.

Due to the negatively charged surface of the clay particles, adsorption of the positively charged low molecular weight organic compounds takes place on the surface of the clay particles by building up ionic bonds.

Depending on the analyte used, the concentration of the analyte in the liquid can vary within a wide range. A person skilled in the art knows that the maximum amount of analyte to be added is limited on the one hand by the solubility of the analyte in the liquid and on the other hand by the fact that at very high analyte concentration saturation of the surface of the clay mineral particles available for the adsorption of the analyte occurs. Furthermore, it was recognized that the concentration to be adsorbed continues to depend on the molecular weight of the analyte to be adsorbed. By the use of the phyllosilicate used according to the invention, it is preferred to achieve absolute adsorbed amounts of analytes of up to 500 mmol / 100 g, more preferably of up to 300 mmol / 100 g, most preferably 200 mmol / 100 g of clay mineral particles.

Preferably, the analyte is present in solution monomolecularly distributed.

According to a preferred embodiment, the analyte is present together with another homogeneously distributed component, wherein the other component, such as the analyte, is selected from the substance group of biomolecules or low molecular weight compounds.

According to a preferred embodiment of the present invention, the contacting of the separating material with the liquid containing at least the analyte takes place over a period of 10 minutes to 24 hours, more preferably 30 minutes to 12 hours.

The contacting of the separating material with the liquid containing at least the analyte preferably takes place at a temperature in the range from 0 to 80 ° C., particularly preferably in the range from 0 to 60 ° C.

The swellable phyllosilicate is preferably selected from the substance group of the two-layer clay minerals (1: 1 phyllosilicates), in particular kaolinite and chrysotile and the three-layer clay minerals (2: 1 phyllosilicates), in particular beidellite, montmorillonite, vermiculite, illite, saponite, smectite , Nontronite, Muscovite, Laponite and Hectorite.

The swellable phyllosilicates are particularly preferably selected from the substance group of the cation-rich 1: 1 phyllosilicates, in particular kaolinite and chrysotile or the cation-rich 2: 1 phyllosilicates, in particular montmorillonite, muscovite, hectorite, laponite and vermiculite. The swellable phyllosilicates are very particularly preferably selected from montmorillonite, hectorite, Laponite (for example Laponite XLG, XLS or RD, RDS) and vermiculite.

The clay mineral particles used according to the invention preferably have a specific surface area in the range from 250 to 2000 m ² / g, more preferably in the range of 500 to 21,000 m / g.

Particularly advantageously, the clay mineral particles used according to the invention have an ion exchange capacity of 30-200 meq / 100 g of clay mineral particles.

Particularly preferred are clay mineral particles having a negatively charged surface and a cation exchange capacity of 30-200 meq / 100 g of clay mineral particles.

According to a preferred embodiment of the present invention, the layers of the clay mineral particles in a range between 0.25 and 2 nm ^{2 are} negatively charged.

According to a preferred embodiment of the present invention, the clay mineral particles are 2: 1 sheet silicates having the general crystal chemical composition [Si ₄ O ₁₀ (OH) ₂ ] ^6- . In addition, cations are incorporated, which are octahedrally surrounded by oxygen atoms. Thus, two octahedral layers are formed on the silicate tetrahedral layer (SiO ₄ ). For preparing the release materials, montmorillonite (Mg _0.33 Al _1.67 [Si ₄ O ₁₀ (OH) ₂ ]) ^x- (Ca, Na) _n (H ₂ O) ^{x +} is preferred, the layer charge x being a number between 0.25 and 0 , 6, or Laponite (synthetic hectorite): Mg _5.34 Li _0.66 [Si ₈ O ₂₀ (OH) ₄ ] Na _0.66 .

The cation-rich 1: 1 layered silicate kaolinite (crystal chemical composition Al ₄ [(OH) ₈ | Si ₄ O ₁₀ ]) or chrysotile (Mg ₃ Si ₂ O ₅ (OH) ₄ ) can equally be used as the clay mineral particles.

The clay mineral particles preferably have a platelet-shaped geometry. The particle diameter of the clay mineral particles is expediently below 2 μm, the clay mineral particles preferably having a height in the range from 0.7 to 2.0 nm.

According to a preferred embodiment of the present invention, the surface of the clay mineral particle used has a surface modification. The surface modification of the clay mineral particles takes place before the synthesis of the release material used according to the invention and is carried out in particular with chlorosilanes, or with methoxy or ethoxy functionalized silanes, which react with the located on the surface of the clay mineral OH groups. Such silanes are known to the person skilled in the art, for example γ-methacryloyloxypropyltriethoxysilane, vinyltrimethoxysilane, 3-aminopropyltriethoxysilane, N- (3-chlorosilylpropyl) dimethylmaleimide, 2- [methoxy (polyethyleneoxy) 6-9propyl] trimethoxysilane.

According to a preferred embodiment of the present invention, the surface modification is carried out with a silane having a volume fraction of not more than 1.0% by volume, more preferably of not more than 0.5% by volume, based on the total volume of the clay mineral particles.

Preferably, the immobilized clay mineral particles are colloidally and homogeneously distributed in the nanocomposite polymer network.

For the purposes of the invention, the clay mineral particles serve as multifunctional crosslinkers for the polymer chains present in the nanocomposite polymer network, whereby a polymer-based gel swellable in water or an organic liquid (in the case of water, a so-called hydrogel) is formed. The effective functionality of the clay mineral particles, i. the ability to bind polymer chains is in the range of 10 to 100, depending on the concentration of the clay mineral particles. For example, a functionality of 10 means that on average 10 polymer arms are bonded, preferably physically, to the surface of a clay mineral particle.

Surprisingly, for the inventors, despite the high functionality (i.e., high level of physically bound polymer concentration) on the surface of the clay mineral particles, an analyte is nevertheless selectively and efficiently absorbed by the nanocomposite polymer network.

Preferred organic polymers in the context of the invention are selected from the group of polyacrylamides, in particular the temperature- and pH-sensitive polyacrylamides (for example poly-N-isopropylacrylamide, N, N'-dimethylacrylamide, N, N'-dimethylaminoethylacrylamide). Furthermore, can be used, inter alia, polyethylene glycols (PEG), polypropylene glycol, and block copolymers thereof (eg. Pluronic ^®), polyvinyl caprolactam, polyvinyl pyrrolidone, polyvinyl pyridine, polyvinyl alcohol, polyoxazolines. The person skilled in the art knows which monomers to take in order to provide the desired polymer.

The organic polymer is particularly preferably selected from the class of the polyacrylamides, in particular poly-N-isopropylacrylamide, N, N'-dimethylacrylamide, N, N'-dimethylaminoethylacrylamide, polyethylene glycol, polyvinylpyrrolidone, polyvinylpyridine, polyvinylcaprolactam, very particular preference is given to the polyacrylamides and / or or polyethylene glycol.

Typically, the organic polymers used in the invention are characterized by a mean Netzkettenmolmasse in the range from 5000 to 2 ⁶ x 10 g mol ^-1, as determined by rheology, from. Particularly preferred according to the invention polymers used have a mean net chain molecular weight in the range of 10,000 to 1 × 10 ⁶ g mol ^-1 .

The person skilled in the art generally knows the methods for the preparation of physically crosslinked nanocomposite polymer networks used in accordance with the invention. For example, nanocomposite polymer networks used according to the invention can be prepared by free radical polymerization in the presence of the abovementioned layer silicates, in particular thermally initiated, in the presence of a free-radical initiator (potassium persulfate (KPS) or AIBN) with or without the addition of a catalyst (for example TEMED) or by photochemical initiation UV light at wavelengths in the range from 200 to 450 nm with a photoinitiator (for example propiophenones or benzophenones, for example 2-hydroxy-1- [4- (2-hydroxyethoxy) -phenyl] -2-methyl-1-propanone) provide. For example Haraguchi et al. (Macromolecules 2002, 35, 10162) and Ferse et al. (Langmuir 2008, 24, 12627) have shown that the polymerization and crosslinking take place in one step. The solvents used are preferably water or hexane.

Alternatively, the nanocomposite polymers used according to the invention can also be provided as so-called shaking gels, in which the polymerization of the monomers is carried out by free-radical or controlled free-radical polymerization (for example via atom transfer radical polymerization (ATRP), nitroxide-mediated radical polymerization (NMRP). or reversible addition-fragmentation chain transfer (RAFT)), and in a second step the resulting polymer solution is mixed with a dispersion of sheet silicates, preferably in water, whereby crosslinking takes place with the sheet silicates ( Ferse et al., Macromol. Symp. 2007, 306-307, 59 and Can et al., Designed Monomers and Polymers 2005, 8, 453 ). Preferably, the average Netzkettenmolmassen the polymers thus obtained are in the range of 10,000 to 500,000 g mol ^-1 .

Surprisingly, the inventors have now found that the nanocomposite polymer networks used according to the invention can also be synthesized via a [2 + 2] cycloaddition of polymer and surface-modified phyllosilicates. The polymerization and crosslinking proceed in two separate steps. For example, in a first step, the polymerization of a copolymer consisting of poly-N-isopropylacrylamide or N, N'-dimethylacrylamide and the acrylamide chromophore N- [2- (3,4-dimethyl-2,5-dioxo-2,5-dihydro -pyrrol-1-yl) -ethyl] -acrylamide by free radical polymerization or by controlled radical polymerization (ATRP, NMRP, RAFT).

For example, the ATRP in a 5: 1 mixture of i-propanol / H ₂ O with the substances CuBr, methyl 2-bromopropionate and the ligand: Me ₆ Tren at temperatures in the Bererich of -10 to 50 °, preferably in the range of 0 to 40 ° C performed. The content of the acrylamide chromophore can be varied in the copolymer in a concentration range of 1 to 15% based on the total amount of the used monomer of the separation material in the synthesis state.

The polymers prepared in this way are reacted with specifically functionalized clay mineral particles of the abovementioned phyllosilicates in the second step. For functionalization, the phyllosilicates are prepared by reaction with various imide-group-functionalized silanes which are generally known to the person skilled in the art (for example N- (3-chlorosilylpropyl) dimethylmaleimide).

Preferably, crosslinking to the nanocomposite polymer networks is by photochemical initiation. The crosslinking is preferably carried out by means of UV light in the range of 200 to 450 nm in order to initiate a [2 + 2] cycloaddition. Sensitizers (e.g., thioxanthone disulfonate) are used for the reactions. Furthermore, crosslinking in a dry state in thin layers is possible.

For the preparation of the release material used according to the invention, the concentration of the clay mineral particles between 0.2 and 20 mol%, based on the total amount of monomer used of the release material in the synthesis state, can preferably be varied.

According to a preferred embodiment of the present invention, the concentration of the polymer in the release material used according to the invention in the range of 3 to 30% by mass based on all components of the release material is set.

The equilibrium swelling ratio with respect to the volume of the hydrogel (Q _V ) is the characteristic basic size of hydrogels, which by definition is calculated as the quotient of the volume of a hydrogel in the swollen state to its volume in the dry state. Upon reaching the magical weight swelling degree, the two counteracting forces, namely, osmotic pressure due to polymer-solvent interactions (swelling) and the repulsive elastic force of the polymeric network are balanced out. The less crosslinker contained in a hydrogel, the larger Q _V and vice versa. Equilibrium swelling levels between 5 and 100 are preferably set in the nanocomposite polymer network used as the release material.

The person skilled in the art is aware that he can freely design the shape and dimensions of the release material depending on its use and the container in which it is introduced. In particular, bulk gels which, in at least one dimension, have a minimum extension of at least 1000 μm are conceivable which, for example, can be brought into the shape of a disk (diskus), a cylinder or a cube.

Of course, the release material may also be formed as a thin film having a layer thickness of at least 50 .mu.m, preferably in the range of 200 to 1000 .mu.m.

Alternatively, the separating material is preferably used as particles having a mean particle diameter in the range from 50 μm to 10 mm, particularly preferably from 200 μm to 5 mm. Particulate separation materials are particularly advantageous since the separation material can be simultaneously flowed around and through liquids, thus providing an enlarged absorption surface in contrast to voluminous or planar separation materials.

According to a preferred embodiment of the present invention, the release material, in particular in the form of a thin film or a bulk gel, is porous. A porous separating material preferably has microchannels with an average diameter in the range from 10 μm to 1 mm, particularly preferably in the range from 20 μm to 500 μm. Preferably, during the synthesis of the nanocomposite polymer network, the microchannels are made by introducing at least one spacer (metal pins of the desired size).

For the purposes of the present invention, typical swelling agents are selected analogously to the abovementioned liquids which contain the analyte or mixtures thereof.

Surprisingly, it has now been found by the inventors that the selective absorption of the analyte in the separation material is reversible, i. that the absorption of the analyte in the separating material by reversing the ambient conditions at any time run in reverse and the analyte can be completely released again.

According to a preferred embodiment of the invention, the analyte is eluted after selective absorption in the release material, in particular in the nanocomposite polymer network by contacting with a solvent. For the purposes of the present invention, elution means the dissolution or release of the analyte bound (absorbed) in the nanocomposite polymer network by the addition of a suitable solvent. In particular, quantitative release takes place by contacting the separation material containing the analyte with a solvent selected from the group of organic surfactants, strong mineral acids (pK _S <3) and strong base (pK _b <3).

Preferably, the solvent for eluting the analyte from the release material is an ionic surfactant, zwitterionic surfactant, in particular 3- [3-cholamidopropyl) -dimethylammonio] -1-propanesulfonate (CHAPS) or nonionic surfactant, in particular from the group of polyoxyethylene sorbitan derivatives (eg . polysorbate 20, Tween ^® 80, Tween ^® X-100), polyalkylene glycol (PEG, polypropylene), Oktylphenolethoxylate (octoxynol 9). More preferably, the solvent CHAPS, octoxynol 9, Tween ^® 80, Tween ^® X-100 or polysorbate 20.

According to a further preferred embodiment of the present invention, the solvent for eluting the analyte from the separating material depending on the dominant surface charge of the clay mineral particle used a strong mineral acid (pK _S <3), in particular hydrochloric acid, sulfuric acid or phosphoric acid or a strong base (pK _b <3), alkali metal hydroxide or alkaline earth metal hydroxide, more preferably NaOH, KOH, Ca (OH) ₂ and Ba (OH) ₂ .

Preferably, the solvent for eluting the analyte is homogeneously dissolved in an aqueous liquid having a concentration of less than 2.0 mol%, more preferably less than 1.0 mol%, most preferably less than 0.5 mol%.

Advantageously, by contacting the separation material containing the absorbed analyte with the solvent to elute the analyte, preferably at the aforementioned concentration, at least 70%, preferably at least 85%, more preferably at least 95% of the absorbed analyte is eluted from the separation material.

According to a preferred embodiment of the present invention, contacting the separation material containing the absorbed analyte several times with fresh solvent to elute the analyte. Preferably, after passage of the solvent to elute the analyte, the various fractions are collected by the separation material.

Particularly advantageously, the separation material can be reused as an absorbent after extraction with water.

According to a preferred embodiment of the invention, the liquid containing the Analytes and / or the solvent for its elution by means of pressure (for example. Apply negative pressure) or centrifugation transported through the release material. This is particularly advantageous if the release material is particulate.

• a) ein Behältnis mit zumindest einem Einlass und einem Auslass,
• b) ein, dem Einlass und dem Auslass zwischengeordnetes, Trennmaterial, welches als vernetztes Nanokompositpolymernetzwerk zum Ionenaustausch ausgebildet ist,
• c) eine Arretierung,

wobei das Trennmaterial unter Standardbedingungen einen Benetzungsfaktor φ größer 0,95 aufweist.The subject of the present invention is also a filter device for the selective separation of an analyte from a liquid comprising at least:

• a) a container with at least one inlet and one outlet,
• b) a separating material interposed between the inlet and the outlet, which is formed as a crosslinked nanocomposite polymer network for ion exchange,
• c) a lock,

wherein the release material under standard conditions has a wetting factor φ greater than 0.95.

In particular, when working under elevated pressure, in particular greater than 200 mbar, it has been found that a lock ( 4 ) of the release material may be advantageous to ensure the fixed positioning of the release material in the container over the entire process for the selective absorption of an analyte. Locking in the context of the present invention means that the release material is physically fixed by a, upstream of the release material or downstream, adhesive element and / or chemically fixed by a bonding agent in the container.

According to a preferred embodiment of the present invention, the surface of a container to be coated or lined with the release material container can be modified by the addition of a coupling agent. The person skilled in the art is aware of adhesion promoters as substances which produce a close physical or mostly chemical bond between the two in the interface of mutually immiscible substances. Depending on the type of material to be coated or lined container, the skilled artisan will recognize which bonding agent he has to use. For example, but not exclusively, the person skilled in the art knows silane-based adhesion promoters, in particular chlorosilanes, or silanes functionalized with methoxy or ethoxy groups, preferably for the modification of glass surfaces, silicone surfaces, amine-based adhesion promoters (in particular for the modification of copper-based or analogous surfaces) or sulfur-based adhesion promoters ( for example, from the group of thiols or sulfides, for example, for the coating of gold).

According to a preferred embodiment of the present invention, an adhesion promoter with a volume fraction of not more than 1.0% by volume, particularly preferably of not more than 0.5% by volume, based on the total volume of all components, is added to modify the surface of the containers.

Liquids in which the coupling agent is present are preferably aprotic solvents, e.g. Bicyclohexyl, toluene, acetone and less preferably ethanol.

In a particular embodiment of the invention is in the container ( 1 ) for receiving the release material in addition to the adhesion promoter or alternatively a downstream of the release material in the flow direction adhesive element, which may be formed, for example. In the form of an open outlet hole blind hole, as a grid or a pin.

According to a particularly preferred embodiment of the invention, the downstream adhesive element is a grid, wherein the mesh size of the grid can be adjusted according to the particle size. The grid preferably has uniformly distributed meshes in the size range from 50 μm to 2000 μm, more preferably between 100 μm and 1000 μm, the values being 200, 300, 400, 500, 600, 700, 800, 900 μm and in each case between the individual ones Size ranges are included. A grid is particularly advantageous if the separating material is particulate, wherein the mesh size can be adjusted according to the particle size of the nanocomposite polymer network.

According to a preferred embodiment of the present invention, the device according to the invention has a porous or at least semipermeable membrane upstream of the separating material in the direction of flow. Advantageously, thereby only separated by size exclusion, homogeneously dissolved in the liquid components and the analyte to the release material, whereas larger components that are present in the liquid, for example, only dispersed, can be retained.

According to a preferred embodiment of the invention, the membrane is porous. The pores of the porous membrane are preferably in the size range from 5 nm to 100 μm, more preferably between 10 nm and 10 μm, the values 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 nm, 1, 2, 3, 4, 5, 6, 7, 8 and 9 μm and the values lying between the individual size ranges are also included.

The skilled person knows that he can freely design the shape of the container for receiving the release material according to the desired purpose and is not subject to any restrictions. Thus, the container can, for example, as a ball, cylinder, cone, truncated cone or cuboid be educated. According to a preferred embodiment of the invention, the material of the container is selected from the group of materials of plastics, glasses or metals / metal alloys.

According to a particularly preferred embodiment of the present invention, the container of the device according to the invention at least one additional inlet and / or outlet, whereby the inflow surface of the separation material for the liquid containing the analyte, or for the solvent is increased and particularly advantageous the mass transfer rate between separating material and liquid is improved.

• a) eine erfindungsgemäße Filtervorrichtung,
• b) einen Auffangbehälter.

The invention also provides a kit comprising at least:

• a) a filter device according to the invention,
• b) a collecting container.

Depending on the desired application purpose, the kit according to the invention preferably already contains a buffer component as defined above, which if appropriate already dissolved in a suitable liquid.

According to a preferred embodiment of the invention, the kit according to the invention has at least one placeholder (for example metal pins of the desired size), which is introduced into the separating material, and advantageously ensures the porosity of the separating material over the period of storage of the kit. The spacer preferably has an average diameter in the range from 10 μm to 1 mm, particularly preferably in the range from 20 μm to 500 μm. It may be provided in particular that the kit has a plurality of placeholders in the separating material, preferably distributed evenly distributed.

According to a preferred embodiment of the present invention, the method according to the invention, the filter device according to the invention and / or the kit for the selective absorption of an analyte in microsystem technology or molecular biology are used.

According to an alternative preferred embodiment of the present invention, the inventive method, the filter device according to the invention and / or the kit are used in the water treatment for the separation of substances from water or wastewater.

LIST OF REFERENCE NUMBERS

1

 container

2

 separating material

3

 outlet

4

 lock

5

 shutter

6

 receptacle

7

 grid

8th

 clay mineral

9

 polymer

10

 liquid

11

 gas inclusion

12

 Bonding / aggregation

13

 eluted analyte in the solvent

14

 solvent

15

 filter means

16

 analyte adsorbed in the release material

embodiments

It should be noted that the embodiments according to the invention can be combined with each other in any arrangement.

Further features and advantages of the present invention will become apparent from the following schematic drawings and exemplary embodiments by means of which the invention is to be explained in more detail by way of example, without limiting the invention to these.

Showing:

1 FIG. 3 shows a schematic cross section through a filter device according to the invention with the separating material (FIG. 2 ) for the selective separation of biomolecules;

2 a perspective exploded view of a filter device according to the invention for the microsystem technology;

3 : a process for the selective separation of biomolecules with the aid of a filter device according to the invention;

4 : The schematic representation of a conventional particle bed (left) in comparison to a nanocomposite polymer network used according to the invention ( 13 ) (right).

1 shows on the left side of a filter device according to the invention ( 15 ) with a release material ( 2 ), which has a lock ( 4 ) in the Bottom area of a container ( 1 ) with an outlet ( 3 ) is introduced and fixed. To distinguish it from external environmental influences, the container ( 1 ) preferably a resealable closure ( 5 ) on. To get out of the outlet ( 3 ) to be able to absorb leaking liquids, the filter device according to the invention in a collecting container ( 6 ), which is formed as a tube arranged. Furthermore, in 1 in the middle section of an alternative embodiment of the filter device according to the invention shown, the bottom portion of a container ( 1 ) with an outlet ( 3 ), wherein the release material ( 2 ) on a lock ( 4 ), which act as grids ( 7 ) is superimposed. On the right side of 1 1 shows a detail of a further embodiment of the filter device according to the invention, wherein the separating material ( 2 ) via a bonding agent with the inner surface of the container ( 1 ) connected is.

In 2 an alternative embodiment of a filter device according to the invention is shown, as it can be used, for example, in microfluidics. Here is the release material ( 2 ) over the entire length of the container ( 1 ), which is used as a tube with outlets ( 3 ) is formed in the jacket, designed. The release material ( 2 ) is via two terminal locks ( 4 ), which are formed as grid elements, fixed in the container. The size of this device can be chosen arbitrarily and adapted to the application.

In 3 schematically a variant of the method according to the invention for the selective separation of biomolecules is shown. In a first step, the according to 1 described device with a liquid ( 10 ), which also contains other components (Δ, •) in addition to the analyte (∎) to be separated. By selective absorption, the analyte in the separating material ( 2 ), wherein the further components with the liquid ( 10 ) from the filter device according to the invention via the outlet ( 3 ) in a collecting container ( 6 ), which is designed as a pool to be discharged. In a final step, the analyte adsorbed in the separating material may ( 16 ) after incubation with a solvent ( 14 ) having a liquid, eluted from the separating material and in a second collecting container ( 6 ), which is designed as a tube to be collected.

4 shows on the left a conventional clay mineral particle-based release material in the form of a particle bed, wherein the clay mineral particles ( 8th ) when sponging in a liquid ( 10 ) with the formation of bonds / clumps ( 12 ) particularly disadvantageous gas inclusions ( 11 ) having. On the right side of 4 is a release material used according to the invention ( 2 ), which serves as nanocomposite polymer network ( 13 ), the clay mineral particles ( 8th ), which have a platelet-shaped geometry, as multifunctional crosslinkers for those in the nanocomposite polymer network ( 13 ) ( 8th ) and thus form a swellable gel which is completely a liquid ( 10 ). Advantageously, the nanocomposite polymer network has no adhesions / lumps ( 12 ) and / or gas inclusions ( 11 ) on.

example 1

In an illustrative experiment, a phosphate-buffered aqueous solution (pH = 7.4) was reacted with the triphenylmethane dyes crystal violet (26 μg / ml, positive charge, 408 g / mole) and cresol red (64 μg / ml, one negative charge; 404 g / mol) was passed over a Polydimethylacrylamid-Laponite gel layer with a diameter of 550 microns. After only 90 minutes, the selective absorption of crystal violet can be clearly recognized. After 6 h, the starting solution is purified to about 95% of crystal violet, while the negatively charged cresol red remains completely in solution. By UV / Vis spectroscopic measurements of the filtered solutions of crystal violet (λ _{max, Ext} = 596 nm) or cresol red (λ _{max, Ext} = 509 nm), these results were confirmed and evaluated.

Example 2:

An E. coli strain (E. coli DH5α) transformed with the plasmid pCMV-Sport6_Bmp4 was dissolved in 500 ml LB medium (1% (w / v) peptone, 0.5% (w / v) yeast extract, 0, 5% (w / v) NaCl, pH 7.4, 100 μg / ml ampicillin) were cultured at 37 ° C for 12 h. The resulting culture was transferred to centrifuge beakers and centrifuged at 5,000 xg and 4 ° C for 15 min to separate the cells. The cells thus obtained were washed twice with Tris buffer (25 mM Tris-HCl, pH 7.5).

The plasmid isolation was carried out according to the manufacturer's instructions of the Macherey nail kit "NucleoBond plasmid purification" for a maxi preparation. The dried DNA pellet obtained after elution and precipitation was dissolved in 2 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and the DNA concentration was determined by microvolume spectrophotometer.

To investigate the absorption behavior of plasmid DNA on a polydimethylacrylamide-montmorillonite nanocomposite, water or a Tris buffer (10 mM Tris-HCl) with different pH values (pH 6.5, 7, 8, 9) was used. The separation material, which was in the form of a block with a mean dimension of 5 mm × 5 mm × 5 mm, was introduced into 1 ml of water or the buffer, with DNA concentrations of 10, 50 and 100 ng / μl being set. The incubation of the samples was carried out for 30 min, 60 min or 90 min in a thermo shaker at 25 ° C. After incubation, the supernatant was removed and the separation material centrifuged for 30 min at 16,000 × g. The concentration of plasmid DNA in the pooled supernatant was determined by microvolume spectrophotometry. The amount of adsorbed plasmid DNA was carried out as a difference determination from the concentration of the plasmid DNA used and remaining in the supernatant after centrifugation DNA amount. Mathematically, after 90 minutes on average, 92% of the amount of DNA originally brought into solution was taken up by the separating material. After 30 minutes of incubation, the average was only 60%.

For the desorption experiments, the supernatant was discarded after centrifugation and the solution remaining in the polydimethylacrylamide-montmorillonite nanocomposite was determined by weighing, whereby the amount of unabsorbed plasmid DNA could also be calculated.

For desorption of the plasmid DNA absorbed in the polydimethylacrylamide-montmorillonite nanocomposite, 2 mg of the substrate were mixed with 1 ml of the desorption buffer (a) water, b) TE buffer, c) phosphate buffer with 0.1% Triton X-100 or Tween 20 or CHAPS mixed. The contacting time was 30 min, 60 min or 90 min at 25 ° C in a thermo shaker. After incubation, the supernatant was removed and the separation material was centrifuged at 16,000 × g for 30 minutes. The concentration of plasmid DNA in the pooled supernatant was determined by microvolume spectrophotometer. After 90 minutes of incubation, water was eluted on average less than 20%, with TE buffer less than 40%, and with phosphate buffer with the added surfactants an average of 94% of the amount of DNA absorbed. After 30 minutes of incubation, the average amounts of DNA eluted were well below the values obtained after 90 minutes.

Example 3:

The absorption and desorption behavior of proteins was investigated using the example of BSA (bovine serum albumin, Roth) or GFP (Green Fluorescent Protein, Institute of Genetics). The proteins were provided at a concentration of 5 mg / ml in MES buffer (20 mM MES, 10 mM NaH ₂ PO ₄ , pH 5.5) and phosphate buffer (10 mM NaH ₂ PO ₄ , pH 7.0, respectively).

To study the absorption behavior of proteins on a polydimethylacrylamide-montmorillonite nanocomposite, water or an MES buffer (20 mM MES, 10 mM NaH ₂ PO ₄ ) with different pH values (pH 5.5, 6.5, 7, 5) used. The release material was polymerized to a thickness of 2 cm in a 50 ml column (ø = 1.5 cm) and overlaid with 10 ml of the buffer, with protein concentrations of 1; 5 and 10.0 mg / ml were set. The incubation of the samples was carried out for 30 min, 60 min or 90 min in a thermo shaker at 25 ° C. After incubation, the samples were centrifuged at 16,000 × g for 30 min and the concentration of protein in the supernatant was determined by microvolume spectrophotometry or by fluorescence measurement. The amount of absorbed protein was determined as difference determination from the concentration of the proteins used and the amount of protein remaining in the supernatant after centrifugation. The average amount of protein absorbed in the release material after 90 minutes was 86% of the amount of protein initially dissolved.

For the desorption experiments, the supernatant was discarded after centrifugation and the solution remaining in the separating material was determined by weighing, whereby the amount of unabsorbed protein could also be calculated.

For desorption of the proteins absorbed in the separating material, the substrate kept in the column was incubated with 10 ml of the desorption buffer a) water, b) MES buffer (20 mM MES, 10 mM NaH ₂ PO ₄ , pH 5.5) or c) phosphate buffer ( 10 mM NaH ₂ PO ₄ , pH 7.0) with 0.1% Triton X-100 or Tween 20 or CHAPS. The contacting time was 30 minutes, 60 minutes and 90 minutes at room temperature. After incubation, the desorption buffer was separated from the separation material by centrifugation within 30 min at 16,000 × g, and the concentration of protein in the supernatant was determined by microvolume spectrophotometer or fluorescence measurement. It was found that by contacting with a surfactant-containing buffer after 90 minutes on average 91% of the amount of protein absorbed could be eluted. After 30 minutes, only less than 38% of the amount of protein absorbed could be recovered. With water, an average of 18% of the amount of protein absorbed was eluted after 90 minutes of incubation.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been 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

• EP 2315815 A1 [0003]
• EP 0937735 A1 [0005]
• US 2014/0158619 A1 [0006]

Cited non-patent literature

• Kaiser et al. (Biochim Biophys Acta 1971, 232 (2), 388-402) [0004]
• DIN 53504 [0017]
• Haraguchi et al. (Macromolecules 2002, 35, 10162) [0053]
• Ferse et al. (Langmuir 2008, 24, 12627) [0053]
• Ferse et al., Macromol. Symp. 2007, 306-307, 59 [0054]
• Can et al., Designed Monomers and Polymers 2005, 8, 453 [0054]

Claims (12)

A method of selectively separating at least one analyte from a liquid comprising the steps of: a) contacting a filter device comprising a release material formed as a nanocomposite polymer network for ion exchange with a fluid containing an analyte and at least one further component, wherein the analyte is attached to the nanocomposite polymer network b) separation of the analyte-free liquid containing at least the one further component from the separating material, characterized in that the wetting factor of the separating material under standard conditions φ is greater than 0.95. A method according to claim 1, characterized in that the analyte after absorption to the release material by contacting with a solvent selected from the group of surfactants, the strong mineral acids and / or the strong bases is eluted. A method according to claim 1 or 2, characterized in that the analyte-containing liquid is passed through the separating material. Method according to one of claims 1 to 3, characterized in that the analyte is monomolecular dissolved. Method according to one of claims 1 to 4, characterized in that the liquid is water or a hydrophilic organic liquid selected from the group of ethanol, methanol, isopropanol, n-propanol, ammonia or acetone or mixtures thereof. Method according to one of claims 1 to 5, characterized in that the nanocomposite polymer network contains phyllosilicates selected from the group of substances of the two-layer clay minerals and three-layer clay minerals. Filter device for the selective separation of an analyte from a liquid, comprising: a) a container with an inlet and an outlet, b) a separating material interposed between the inlet and the outlet, which is formed as a crosslinked nanocomposite polymer network for ion exchange, c) a lock, wherein the release material under standard conditions has a wetting factor φ greater than 0.95. Filter device according to claim 7, characterized in that the separating material in the flow direction is preceded by a porous or at least semipermeable membrane. Filter device according to claim 7 or 8, characterized in that the lock is a bonding agent or an adhesive element. Kit comprising at least: a) a filter device according to one of the preceding claims, b) a collecting container. Use of a method according to one of the preceding claims, a filter device according to one of claims 7 to 9 and / or a kit according to claim 10 for the selective absorption of an analyte in microsystem technology or molecular biology. Use of a method according to one of the preceding claims, a filter device according to one of claims 7 to 9 and / or a kit according to claim 10 in the water treatment.

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