DE29913649U1 - Novel template-embossed materials - Google Patents

Description

The invention relates to novel template-embossed materials in the form of template-embossed polymers (TGP) on any surface, including membranes (template-embossed membranes, TGM). They are created by modifying the surface of solid supports, which leads to stable template imprints by means of a cross-linking &iacgr;&ogr; polymerization of functional monomers initiated on the support surface in the presence of a template, which then specifically bind template molecules or template derivatives from organic or aqueous, saline solutions.

In biotechnology, new, efficient separation and purification strategies are required for products such as enzymes, monoclonal antibodies or recombinant proteins. This also applies to synthetic active ingredients, especially if they have a more complex structure and/or a higher molecular weight or limited stability.
For all of these areas of application, substance-specific high-performance materials are sought, whereby a high degree of flexibility in adaptation to the specific targets is required. Preferably, solid materials (particles, films) are used to simplify the phase separation of solid and liquid material streams. In contrast to separation processes that are based on different physical properties, chemical affinity to the carrier is the prerequisite for substance-specific separations. Substance specificity can be achieved by biological or biomimetic receptors. To date, either specific but very sensitive biological ligands (e.g. antibodies, enzymes) or relatively unspecific synthetic ligands (e.g. dyes, metal chelates) have been used for affinity separations; examples are chromatography, solid phase extraction, membrane separation, solid phase assays or sensors (D. Sii, A. Sadana, J. Biotechnol. 19 (1991) 83).

Pore-free films or layers or particles with affine ligands on the surface have a limited binding capacity; porous materials with a larger specific surface area and binding capacity typically have limitations in binding capacity due to diffusion limitations. Directed flow-through porous membranes are therefore particularly attractive alternative materials. Established membrane processes with porous membranes such as micro- or ultrafiltration (MF or UF) function according to the size exclusion principle (W. Ho, K. Sirkar (Eds.), Membrane Handbook, van Nostrand Reinhold, New York, 1992). The separation of substances of similar molecular size with porous membranes also requires specific (affinity) interactions with the membrane (E. Klein, Affinity Membranes, John Wiley & Sons, New York, 1991).

The main motivation for the use of affinity membranes is the possibility of directed flow to separation-specific groups (ligands/receptors) that are located in high density in the pores. This allows a drastic improvement in efficiency (lower pressure drop, shorter residence times, higher

Throughput rates, hardly any diffusion limitation in pores, faster equilibration) are possible compared to analogous processes with particles (DK Roper, EN Lightfoot, J. Chromatogr. A 702 (1995) 3). Such affinity membranes can be used for substance separation, preferably of proteins, but also of many other substances (e.g. peptides, nucleic acid derivatives, carbohydrates or various toxins, herbicides, pesticides) up to cells (US 5766908). In addition, there are a wide range of possible applications in analytics, such as for highly selective sample enrichment or for decontamination of material flows (DE 19609479).

A very attractive alternative** to biological or biomimetic affinity ligands/receptors, e.g. for chromatography or analytics, has been developed in recent years. This is the use of specific but very robust functional cavities ("molecular imprints") in synthetic polymers, produced by molecular imprinting polymerization (G. Wulff, Angew. Chem, 107 (1995) 1958; K. Mosbach, O. Ramström, Bio/Technology 14 (1996) 163; AG Mayes, K. Mosbach, Trends Anal. Chem. 16 (1997) 321). This involves polymerizing monomers in the presence of template molecules (e.g. protein, nucleic acid, low molecular weight organic substance) which, with a functional monomer, form a relatively stable

γ complex. After washing out the template, the materials produced in this way can specifically bind template molecules again. The polymers synthesized in this way are called template-imprinted polymers (TGP) / molecularly imprinted polymers (MIP) or "fingerprint" polymers (see Fig. 1, Fig . T).

In this way, for example, the production of polymeric sorbents in the presence of small

The synthesis of organic molecules (patent US 5110833) or macromolecular substances (US 5372719) or the synthesis of acrylamide or agarose gels in the presence of proteins have been described (patents US 5728296, US 5756717). Peptide or protein-specific sorbents, produced by "surface imprinting" of metal chelate structures on specially functionalized particles, have also been described (US 5786428). In all cases, high affinities for the respective templates were obtained.

The use of artificial antibodies and receptors produced by molecular imprinting has great advantages because these structures are much more stable than their natural analogues. In addition, they can be synthesized for any substance (even those with weak antigenic properties, such as small molecules or immunodepressants) and can be produced much more easily and cheaply than the corresponding biomolecules.

The selection of components for the synthesis of a TGP is primarily based on the interactions between the template and the functional monomer. With the aim of "fixing" these interactions as effectively as possible and making them accessible for affinity interactions, suitable cross-linkers and solvents are also selected.

Any substance with a defined three-dimensional shape can be used as a template for the synthesis of TGP . Substance classes therefore range from small molecules with molecular masses below or around 100 Da (e.g. herbicides) to particles such as viruses, bacteria or cells. However, compounds with biological functions such as proteins, peptides, nucleic acids or carbohydrates are of particular interest. The recognition of templates by TGP is based on the combination of various factors such as reversible covalent or non-covalent bonding, electrostatic and hydrophobic interactions and the complementarity of the shape. Which of these factors dominates depends on the polymer structure, the template properties and the binding conditions. For example, in hydrophobic solvents, electrostatic interactions are often dominant for template recognition by TGP . In polar solvents, however, hydrophobic interactions and shape specificity are most important for template recognition. Preferably, TGP should be synthesized under conditions that favor strong but reversible interactions between the polymer and the template. For large molecules (100...100,000 Da), however, a combination of many weaker bonds including hydrogen bonds and hydrophobic interactions can be favorable. For small molecules (50...100 Da), a few strong interactions such as ionic bonds are necessary to obtain TGP with high affinity.

Water as a solvent or aqueous systems in general are of particular interest in connection with the above-mentioned applications. "Optimized" by nature

Ligand/receptor systems function optimally under these conditions. In contrast, the synthesis of TGP for applications in aqueous systems has only recently seen initial progress (L. Andersson, Anal. Chem. 68 (1996) 111; S. Hjerten, JL Liao, K. Nakazato, Y. Wang, G. Zamaratskaia, HX Zhang, Chromatographia 44 (1997) 227). The synthesis of T^P receptors for smaller molecules poses particular problems. Evidently, such attempts have so far only partially succeeded in controlling not only the choice of suitable interaction forces but also the detailed arrangement of the functional groups.

The invention is based on the task of improving the known state of the art by developing

The aim was to improve the properties of template-imprinted materials. The tasks were solved by synthesizing new types of template-imprinted polymers (TGP), eg on the surfaces of solid molded bodies as carriers.

The present invention comprises a general polymerization process; in a manner known in principle, under the specific conditions according to the invention (see below), the

The synthesis of TGP particles is possible. However, since these particles have a more or less strong hydrogel character due to the preferred water-soluble monomers and aqueous conditions (see below), the synthesis of thin TGP layers on solid, preferably polymeric, supports is preferably carried out. This surface imprinting leads to covalently fixed, thin layers with template imprints on the entire support surface, for example a membrane (TGM), through a controlled cross-linking polymerization initiated selectively on the support surface in the presence of template molecules. Due to the selectivity of the initiation, the matrix or pore structure remains intact. This means that an independent optimization of pore structure (capacity, permeability) and surface functionality (specificity through template imprints) can be achieved using a special manufacturing process. Possible templates are, for example, small molecules with molecular masses below or around 100 Da (including herbicides), larger molecules such as peptides, proteins, nucleic acids or carbohydrates, or even particles such as viruses, bacteria or cells. When filtered through or applied to TGP, the templates or template derivatives can also be bound from diluted solutions in the template imprints (functional cavities) with high specificity. The templates or template derivatives can then be purified if necessary and subsequently either eluted under filtration conditions (as a concentrate) or detected directly on the carrier.

Polymeric membranes can be produced in a variety of pore structures and with the desired mechanical etc. properties by processes such as precipitant or temperature-induced phase inversion. This allows a selection of optimal porous matrix membranes for the desired separation processes (E. Klein, Affinity Membranes, John Wiley & Sons, New York, 1991).

The selection of components for a template-specific TGP is primarily based on the interactions between template (T) and functional monomer (FM). With the aim of "fixing" these interactions as effectively as possible and making them accessible for affinity interactions, suitable cross-linkers (V) and solvents (LM) are selected. The synthesis of the 7Y7P layers is then carried out in situ by reactive coating of the entire carrier surface, e.g. membrane, from a reaction mixture of low viscosity, but with retention of the complex of T and FM. This functionalization of the membrane with TGP is carried out in such a way that neither the pore structure nor the stability of the matrix membrane are impaired, but that blocking of the (transmembrane) pores is also minimized.

In order to meet these requirements, photochemical initiation of a heterogeneous graft copolymerization (e.g. functional acrylates) for TGP synthesis is particularly preferred. This results in the following basic functionalization sequence:

1. Coating the carrier with photoinitiator (PhF)

2. Equilibration of the carrier with the reaction mixture (T, FM, V, LM, PhT),

in the case of membranes: filling and equilibrating the pore volume of the matrix membrane with the reaction mixture,

3. UV exposure: selective excitation of the PhI, generation of starting radicals on the surface of the carrier, polymerization (preferably temperature, T < 25 ⁰ C),

4. Extraction of unreacted reactants, soluble homopolymer and T.

These functionalizations are based on the effect of the carrier material as a coinitiator, ie all polymers from which photoinitiated radicals can be generated that can start a graft copolymerization can be modified in this way. The TGP

Functionalizations are possible from aqueous or organic solvents. The degree of functionalization and thus the surface coverage of the matrix can be controlled via initiation and polymerization conditions. If necessary, blockage of the matrix membrane pores can also be minimized in the case of TGM . This allows the application of the method arsenal established for TCP synthesis to surface functionalization.

is possible from carrier materials.

For photofunctionalization, a previously unknown interaction between the adsorption of PhI and T on the (membrane) polymer surface and the polymerization conditions was observed. An adsorbed hydrophobic PhI (e.g. benzophenone) can be removed from a hydrophilic polymer (e.g. nylon) by a hydrophilic T (e.g.

terbumetone); photofunctionalization is thereby suppressed. Such a system is not suitable for JGP synthesis. A hydrophilic PhI (e.g. benzophenonecarboxylic acid), on the other hand, coadsorbs with the hydrophilic T on the hydrophilic polymer; photofunctionalization is thus sufficiently effective; the T binds specifically to the TGP synthesized in this way. A hydrophobic PhI (e.g. benzophenone) preferentially adsorbs on a hydrophobic polymer (e.g. polypropylene); photofunctionalization is effective; for a more hydrophilic T (e.g. terbumetone), the bonds to template imprints in the graft copolymer dominate. Different rCP structure types can be derived from this (see Fig. 3):

a) template imprints within and/or on the surface of a cross-linked graft copolymer layer fixed to the carrier surface,

b) Template imprints directly on the carrier surface with the involvement of the matrix polymer.

Chemical grafting of polymers or polymer cross-linking (e.g. synthesis of polyaniline derivatives) on the surface of a matrix membrane is also suitable for the production of TGP .

Template. Suitable substance classes range from small molecules with molecular masses below or around 100 Da (e.g. herbicides) to particles such as viruses, bacteria or cells. The template concentrations in the monomer mixture for TCP production are between 0.01 and 50%. With the present invention, ionic and electrostatic interactions as well as hydrogen bonds can also be used in aqueous systems for the synthesis of TGP and thus for molecular recognition. Hydrophobic interactions can make an additional contribution. This results in significant improvements, especially for small molecules, and can also be used for biologically relevant molecules such as amino acids, peptides, nucleic acids, oligonucleotides, sugars and oligosaccharides, but also for proteins or DNA and RNA.

Functional monomers with positively or negatively charged functional groups (e.g. aminoethyl acrylate derivatives or acrylic acid or methacrylic acid) are suitable for TCP synthesis. In addition, hydrophobic units such as aromatic rings, cryptands or cyclodextrins can be incorporated into TGP . Monomers capable of complex formation such as metal chelate complexes, Schiff bases and special esters can also be used for the

used to produce TGP . Aniline and its derivatives with other functional

Groups can be used for the rCrP synthesis. Furthermore, derivatives of phenylboronic acid, which can form esters with diols, are also suitable as functional monomers. The concentration of functional monomers in the mixture can be between 0 and 100%. The solvents for the polymer production can be the monomer itself, water (buffer), organic solvents or mixtures thereof. In general, the optimal monomer type for TGP depends on the template structure and the polymerization conditions. Depending on the polymerization conditions and the composition, the embossed polymers can be produced in the desired density, porosity, crosslinking density and consistency. Examples of crosslinkers are ethylene glycol bismethacrylate for functional

acrylates, o-phenylenediamine for polyaniline, �N,�N-methylenebisacrylamide or piperazinebisacrylamide for acrylamide or bisepoxides for agarose. The crosslinker concentrations in the monomer mixture are between 0 and 80%.

To wash the template out of the TGP , an acid that disrupts the electrostatic interactions, a salt solution with an ionic strength sufficient for dissociation, or a solvent with a different polarity can be used. This releases the binding sites complementary to the template structure in the pores and/or on the surface of the polymer. However, applications of TGP with bound template are also possible.

By choosing the functionalization strategy or conditions, not only can the template imprints be optimally specific, but also the non-specific interactions of structurally similar or different substances with the TGP be minimized. Examples of this are the optimization of the functional monomer and/or the crosslinker, additives in the reaction mixture (e.g. hydrophilic monomers), multi-step modifications or subsequent derivatization. This increases the selectivity of the TGP .

The structural characterization of the TGP is carried out in a basically known manner using established methods, e.g. by SEM investigations (SEM - scanning electron microscope), FTIR-ATR spectroscopy (Fourier Transform Infrared - Attenuation of Total Reflection; infrared spectroscopy with attenuation of total reflection), functional group analysis using photometric or fluorometric methods, contact angle measurements, permeability measurements and static and dynamic sorption experiments with the template or other structurally similar or different substances. In particular, the static and dynamic binding capacities of the TGP for the template, depending on the rGP structure and the test conditions (concentration, residence time, applied amounts and volumes of substances, rinsing conditions, etc.) are essential with regard to the diverse applications of the TGP.

Templates or template derivatives are bound with high specificity in the template prints during filtration through or application to TGP, even from high dilution. The templates or template derivatives can then be purified if necessary and subsequently either eluted under filtration conditions (as a concentrate) or detected directly on the carrier (see Fig. 4).

The following applications result from the solution according to the invention, without limiting the possible applications to the specific cases:
1. Filtration (perfusion) of solutions, but also gaseous mixtures, by TGP,
2. Diffusion (dialysis) or electrodiffusion (dialysis) by TGP,

3. Application of TGP in solid phase extraction, (membrane) chromatography or electrophoresis,

4. Application of TGP in sensors,

5. Application of TGP as a catalyst,

see also 6: Application of solutions, but also gaseous mixtures, to TGP; examples: test strips, blotting membranes, assays, drug screening.

With the synthesized TGP, for example, the efficient and specific concentration of pollutants (herbicides) from diluted solutions is possible (see L). On the one hand, this can be used to quantitatively eliminate such substances (detoxification); on the other hand, however, a defined enrichment (analogous to solid phase extraction; see 3.) is also possible for the subsequent trace analysis. For example, the efficient purification of proteins, a process of the highest relevance for biotechnology, is also possible with TGP (see 1). Here too, depending on the protein and process, both analysis, production in pure form and decontamination are possible.

According to the invention, the known state of the art can be improved by novel TGPs which are synthesized from aqueous reaction mixtures and also exhibit high specificities under aqueous conditions, in particular in the presence of buffer salts.

A complex between template and functional monomer, which is essentially based on ionic bonds, can be easily cleaved by increasing salt concentration. This effect can be used for the elution of the template from the TGP , but it severely limits the applicability for affinity separations, with the exception of aqueous solutions of low ionic strength.

Surprisingly, the addition of salt (e.g. buffer) during polymerization leads to TGPs that have a high affinity for template molecules at a similarly high salt concentration (e.g. of the buffer under the preferred application conditions). This effect can be described phenomenologically in such a way that the salt concentration in the reaction mixture "sets" the distance between the functional groups involved in the ion exchange interaction. In the course of the synthesis, it is evidently possible to fix this advantageous constellation (synthetic receptor with ionic functional groups at the correct distance). This procedure can be implemented particularly effectively for surface functionalization of solid supports, which is described below.

By choosing the functionalization strategy or conditions, not only can the template imprints be optimally specific, but also the non-specific interactions of structurally similar or different substances with the TGP be minimized. Examples of this are the optimization of the functional monomer and/or the crosslinker, additives in the reaction mixture (e.g. hydrophilic monomers), multi-step modifications or subsequent derivatization. This increases the selectivity of the TGP .

The novel template-imprinted materials according to the invention consist of template-imprinted polymers (TGP) which are obtained by modifying the surface of solid supports in aqueous or organic reaction solutions and which lead to stable template imprints by means of a cross-linking polymerization of functional monomers initiated on the support surface in the presence of a template. The template imprints can then specifically bind template molecules or template derivatives even from aqueous, salt-containing solutions.

The template-imprinted membranes (TGM) according to the invention are obtained by modifying the surface of membranes, which leads to stable receptor structures in the form of template imprints by means of a cross-linking polymerization of functional monomers initiated on the membrane surface in the presence of a template, which can then specifically bind template molecules or template derivatives.

Organic polymers such as polypropylene, polyethylene, polystyrene, polysulfone, polyamides, polyesters, polycarbonate, polyacrylonitrile, polyvinylidene fluoride, polytetrafluoroethylene, polyacrylates, polyacrylamides, cellulose, amylose, agarose and their respective derivatives, copolymers or blends of the polymers serve as carrier materials.

Porous solids such as glasses, silicates, ceramics or metals or their composites, also with organic polymers, are also used as carrier materials.
!"*. J·** .**. .··. .··. .: ·*·; .·· t .··. : : ·

The membranes preferably have symmetrical, but also asymmetrical pore structures and pore sizes between a few nm and 10 μηη, preferably 100 nm to 5 μηη.

Small molecules with molecular masses below or around 100Da, such as herbicides, active ingredients or amino acids, larger molecules such as peptides, proteins, nucleic acids or carbohydrates, or particles such as viruses, bacteria or cells, and functional monomers are polymerizable compounds with groups capable of interacting with templates, in particular carboxyl, sulfonyl, sulfate, phosphate, amino or quaternary ammonium groups and their derivatives, also in mixtures.
&iacgr;&ogr; Subsequent or prior additional functionalization or coating reduces the nonspecific binding of template-competing substances and non-templates.
In a special embodiment, in a mixture with the functional monomers

also crosslinking monomers and solvents for all components of the
is used in the reaction mixture.

In the production of template-imprinted polymers (TGP), the binding specificity and capacity of the template-imprinted polymer for the template and template-like substances can be increased by the addition of salt. Films, membranes, fibers, hollow fibers, fabrics, nonwovens or particles, each nonporous or porous, are used as carriers, but the template-imprinted polymer can also be produced without a carrier in any shape and size.

In the production of template-embossed materials according to the invention, a substance of the H-abstraction type (addition of a hydrogen atom from the environment) is used as a photoinitiator and the carrier polymer as a coinitiator, whereby the initiation takes place by light excitation of the photoinitiator.

The materials according to the invention are suitable for the separation and/or analysis of

liquid or gaseous mixtures based on the specific binding of the template
or template derivatives in perfusion or diffusion through template-imprinted polymers or application to template-imprinted polymers, furthermore in the
substance-specific separation using

- Affinity filtration through a template-imprinted polymer arrangement for
Concentration, purification, separation or analytical determination of substances,

- Dialysis or electrodialysis through a template-imprinted material for concentration,
Purification, separation or analytical determination of substances,

- Solid phase extraction, chromatography, membrane chromatography or electrophoresis with a template-imprinted polymer for the concentration, purification, separation or analytical determination of substances,

- a template-imprinted polymer as a sensor or catalyst for the purification, separation or analytical determination of substances and

- a template-imprinted polymer as a blotting membrane or test strip or material for assays or for drug screening.

The features of the invention are not only evident from the claims but also from the description, whereby the individual features, either individually or in combination, represent advantageous, protectable embodiments for which this document

Protection is applied for. The combination consists of known (membranes, polymers) and new elements (modification of the entire surface of membranes with template-embossed polymer layers, synthesis of thin TGP layers on solid supports), which influence each other and in their new overall effect result in a practical advantage and the desired success, which lies in the fact that now with the synthesized TGP , for example, the specific concentration of pollutants from diluted solutions (enrichment of trace analysis) as well as the efficient high-purification of proteins - a process of the highest relevance for biotechnology - is possible and the TGP synthesis in aqueous systems with high template specificity as well as the applicability of TCP synthesis to

&iacgr;&ogr; surfaces of solid molded bodies as carriers succeeds.

The invention will be explained in more detail using exemplary embodiments, without being limited to these examples.

Examples of implementation

Example 1. Molecularly imprinted polypropylene membrane for the herbicide terbumetone (2-t-butylamino-4-ethyl-6-methoxy-I,3,5-triazine)

A sample (6 cm ² ) of a PP-MF membrane (2E HF, Akzo Nobel, Wuppertal) is extracted with chloroform, dried and weighed; the membrane is then soaked in a Petri dish (d=10 cm) with 10 ml of a reaction solution consisting of 5 mM terbumetone (template), 25 mM acrylic acid (functional monomer), 600 mM ethylene glycol bismethacrylate (crosslinker) and 5 mM benzophenone (photoinitiator) in chloroform and covered with a layer. The Petri dish is covered with a glass plate (deep UV filter, 1> 310 nm). After 30 min, the membrane is exposed at half load on a UV dryer (Beltron GmbH) for a total of 9 min (9 passages through the exposure zone). The membrane is then extracted three times with chloroform/acetic acid (98/2, v/v) and three times with chloroform. Afterwards, the sample is dried and the degree of modification, TGP per outer membrane area, is determined gravimetrically: DG = 3.3 μg/cm ² .

A non-molecularly imprinted control sample is prepared according to the same procedure, but without template; the degree of modification (polymer per outer membrane area) is:

Example 2. Molecularly imprinted nylon membrane for the herbicide terbumetone (2-t-butylamino-4-ethyl-6-methoxy-l,3,5-triazine)

A sample (6 cm ² ) of a Ny-MF membrane (2D, Akzo Nobel, Wuppertal) is extracted with chloroform, dried and weighed; then the membrane is soaked in a Petri dish (d = 10 cm) with 10 ml of a reaction solution consisting of 1 mM terbumetone (template), 5 mM acrylic acid (functional monomer), 120 mM ethylene glycol bismethacrylate (crosslinker) and 1 mM benzophenone-4-carboxylic acid (photoinitiator) in chloroform and covered.

The Petri dish is covered with a glass plate (deep UV filter, l>310nm). After 30 min, exposure is carried out at half load on a UV dryer (Beltron GmbH) for a total of 9 min (9 passages through the exposure zone). The membrane is then extracted three times with chloroform/acetic acid (98/2, v/v) and three times with chloroform. It is then dried and the degree of modification (TGP per external

membrane area): DG = 24.2 μg/cm ^z .

A non-molecularly imprinted control sample is prepared according to the same procedure, but without template, the degree of modification (polymer per outer membrane area) is:

DG = 30.0 μg/cm ² .

Further results for varied preparation conditions are shown in Table 1.

Table 1: Preparation conditions and results for the herbicide terbumetone
Polypropylene membranes (PP) and nylon membranes (PA)
Initiator: 1 mM benzophenone; * 1 niM benzophenone-4-carboxylic acid

membrane Terbumetone
(mM) A.A.
(mM) EDMA
(mM) DG
^g/cm ² ) PP-MIPl
PP-Cl 0.2 1 24 0
0 PP-MIP2
PP-K2 1 5 120 2.0
3.3 PP-MIP3
PP-K3 5 25 600 7.5
7.7 &Rgr;&Rgr;-&Mgr;&Ggr;&Rgr;4*
PP-K4* 1 5 120 0
3.3 PA-MIPl
PA-Cl 0.2 1 24 0'
13.2 &Rgr;&Agr;-&Mgr;&Ggr;&Rgr;2
PA-K2 1 5 120 1.7
17.6 &Rgr;&Agr;-&Mgr;&Ggr;&Rgr;3
PA-K3 5 25 600 0
0 PA-MJP4*
PA-K4* 1 5 120 24.2
30.8

MIP molecularly imprinted membrane,

K non-molecularly imprinted membrane (control),

AA Acrylic acid,

EDMA ethylene glycol bismethacrylate,

DG Modification Degree

Example 3. Application of a polypropylene membrane molecularly imprinted for terbumetone for the enrichment of herbicides (solid phase extraction)

A sample (4.9 cm ) of a membrane modified according to Example 1 is mounted in a steel filter holder with a Luer-Lock connection (Schleicher & Schuell, Dassel). 10 ml of a solution of the herbicide (terbumetone, terbutryn, desmetryn or terbutylazine) with a concentration in the range of 10'7 to 10~5 M in water are quantitatively filtered through the membrane from a syringe at a rate of 10 ml/min. The filtrate and 10 ml of the crude solution are then extracted with 10 ml of chloroform each; the herbicide concentrations are then determined using gas chromatography (separation column HP5MS; Hewlet Packard GC System HP 6890 with mass-selective detector HP 5973).

quantitatively and thus determines the amount bound in the membrane.

Representative results for PP-MIP, PP-control and PP-unmodified as well as 5* 10" ⁷ M herbicide in water are shown in Fig. 5 and Table 2.

Table 2: Adsorption capacity of a molecularly imprinted PP membrane
(10 ml herbicide solution; concentration 10~5 M; membrane area 5 cm ² )

membrane Terbumetone nmol/cm^ adsorption Terbutylazine % nmol/cm^ Desmetryn % nmol/cm^ Terbutryn % nmol/cm^ % 15.6 95 19.0 39 7.8 98 19.6 78 15.8 95 19.0 42 8.4 71 - 14.2 PP 79 19.2 76 15.2 63 12.6 100 20.0 PP-K3 96 PP MP3

PP unmodified membrane,

PP-K3 membrane modified without the presence of template,

PP-MIP3 molecularly imprinted membrane (T: Terbumetone)

Example 4. Peroxidase embossed polypropylene membrane

Aniline is polymerized on a polypropylene membrane to form a firmly adhering, homogeneous, optically transparent film according to the following exemplary procedure (see Figure 1): 20 μl of ammonium peroxodisulfate (250 mM in water) are pipetted into 30 μl of a solution of aniline hydrochloride (720 mM) and horseradish peroxidase (1.67 mg/ml) in water, mixed thoroughly and allowed to react for 2 hours at room temperature while shaking. The mixture is then washed thoroughly with water and then with 10 mM sodium phosphate buffer (pH = 7.5).

Example 5: Replacement of biological receptors in assays by molecularly imprinted polymer membranes

Membranes were modified with peroxidase-TGP as in Example 4. The modified surfaces obtained show the behavior of artificial antibodies for peroxidase. To demonstrate the affinity of the TGP surfaces for the template peroxidase, horseradish peroxidase is adsorbed from solutions of different concentrations and then determined using hydrogen peroxide addition and Vis detection based on the sensitivity of the PAni film. The significantly higher values for the peroxidase-TGP surface compared to the very low signal in the non-imprinted control sample show the higher binding of horseradish peroxidase to the synthetic receptor structures with saturation of the sorption capacity in the concentration range investigated.

Example 6. Molecularly imprinted polypropylene membranes for the herbicide Desmetryn (l-isopropylamino^-methylamino-o-methylthio-1,3,5-triazine)

A round sample (46 cm ² ) of a PP-MF membrane (nominal pore size, d = 0.2 μηι; 2E HF, Akzo Nobel, or d = 0.6 μηι; AN 06, Millipore) is extracted with chloroform and methanol, dried and weighed. The membrane is then immersed in a 100 mM

Solution of BP (photoinitiator) in methanol. The membrane, whose pores are still filled with the BP solution, is then placed in a Petri dish (d = 10 cm) and covered with 20 ml of reaction solution consisting of 10 mM desmetryn (template), 50 raM AMPS (functional monomer), 100 mM MBAA (crosslinker) and 0.1 mM BP in water. The Petri dish is covered with a glass plate (deep UV filter, 1 > 310 nm). After 30 minutes, the membrane is exposed to light in a UV dryer (Beltron GmbH) at half load for a total of 10 minutes (10 passes through the exposure zone). The membrane is then washed intensively with methanol, water, 50 mM hydrochloric acid, water and methanol again. It is then dried and the degree of modification (DG, based on the outer membrane surface) is determined gravimetrically. A non-molecularly imprinted control sample is prepared according to the same procedure, but without template. Results for varied preparation conditions (pH value, salt concentration) are shown in Table 3.

Example 7. Application of Desmetryn-embossed polypropylene membranes for the enrichment of herbicides (solid phase extraction)

A sample (4.9 cm ² ) of a membrane functionalized according to Example 6 (see Table 3) is mounted in a steel filter holder with a Luer-Lock connection (Schleicher & Schuell, Dassel). 10 ml of a solution of desmetryn with a concentration in the range of 10" ⁷ to 10" ⁵ M in water are quantitatively filtered through the membrane from a syringe at a rate of 10 ml/min. The filtrate and 10 ml of the crude solution are then extracted with 10 ml of chloroform each; the herbicide concentrations are then quantitatively determined using gas chromatography (separation column HP5MS; Hewlet Packard GC System HP 6890 with mass-selective detector HP 5973) and in this way the amount bound in the membrane is determined.

Representative results are summarized in Table 4. For both matrix membrane pore sizes, the rGP membranes show significantly (TGP 1) or significantly (TGP 2) higher affinities for the herbicide, while the membranes modified with a chemically similar but non-imprinted polymer show slightly increased (K 1) or even lower (K 2) values compared to unmodified PP.

The TCP-bound herbicide can be eluted from the membrane by changing the pH or increasing the salt concentration; an example is shown in Fig. 6.

In a basically analogous manner, the herbicide could be enriched 1000-fold from a 2*10'9m solution with a recovery of 90%; i.e. a substance-specific solid phase extraction can be used both for purification and concentration.

The TCP membranes can be used repeatedly without loss of specificity and capacity after a simple regeneration, ie an application for decontamination is possible (see Fig. 7).

Example 8. Substance or group specificity of desmetryn-imprinted polypropylene membranes

A sample (4.9 cm2) of a membrane functionalized according to Example 6 (TGP 1, see Table 3) is tested as described in Example 7: 10 ml of a solution of the respective herbicide (desmetryn, terbumetone, terbutryn or terbutylazine) with a concentration of 10 ^-5 M in water are quantitatively filtered through the membrane from a syringe at a rate of 10 ml/min. The quantitative analysis is then carried out as described in Example 7.

Statements on the specificity of imprinting and the group specificity of the JGP membranes can be derived from the results shown in Fig. 8.

For the template desmetryn as well as terbutryn and terbumetone (methoxy- and methylthio-substituted s-triazines, respectively), the affinity of TGP is greater than that of K. For terbutylazine (chlorine-substituted s-triazine) and the 1,2,4-triazine metribuzin, however, no specificity can be observed. This means that the specificity is generated by imprinting; even substances with identical hydrophilicity/phobicity balance (terbumetone and terbutylazine, each lgk _ow = 3.04) are bound due to a different molecular detail.

(methoxy vs. chlorine substituent) are bound differently by the synthetic receptor γ ("Fit"). This results in a group specificity for s-triazines with similar substitution ("polyclonality", in analogy to antibodies).

Example 9. Affinity of different Desmetryn-impregnated polypropylene membranes for herbicides from aqueous buffer solutions

A sample (4.9 cm^) of a membrane functionalized according to Example 6 (see Table 3) is tested as described in Example 7: 10 ml of a solution of desmetryn with a concentration of 10~5 M in buffer solutions with different pH values are quantitatively filtered through the membrane from a syringe at a rate of 10 ml/min. The quantitative analysis is then carried out as described in Example 7 (see Fig.

It can be seen that the TCP membranes polymerized from salt-free solutions (see Table 3) have a low affinity for herbicide binding from buffer solutions. Synthesis at pH = 5.5, ie with the sodium salt of the functional monomer AMPS, also results in similarly low affinities. In contrast , TGPs synthesized at low pH with the addition of salt show remarkably high affinities for herbicide binding from 50 mM buffer solutions. At higher pH values, the affinity of the TGPs decreases, obviously because salt formation increasingly competes with the binding of the herbicide in the synthetic receptor.

Example 10. Replacement of biological receptors (here anti-atrazine antibodies) in assays by TGP -Surfaces

Wells of 96-well polystyrene microtiter plates were modified under analogous conditions as described in Example 6, but with atrazine as template:

First, 250 μl of a 100 mM solution of BP (photoinitiator) in methanol are pipetted into each well and the microtiter plate is shaken for 60 min. The solution is removed and then 250 μl of the reaction solution consisting of 10 mM atrazine (template), 50 mM AMPS (functional monomer), 100 mM MBAA (crosslinker) and 0.1 mM BP in water are pipetted into each well. The microtiter plate is covered with a glass plate (deep UV filter, 1 > 310 μm). After 60 min, exposure is carried out on a UV dryer (Beltron GmbH) at half load for a total of 10 min (10 passages through the exposure zone). The microtiter plate is then washed intensively with methanol, water, 50 mM hydrochloric acid, water and methanol again and then dried.

The modified surfaces of the wells obtained in this way show the behavior of artificial antibodies for atrazine, which can be used in a competitive triazine assay:

In different modified wells, 50 μl of a solution of herbicide (atrazine or metribuzin) in concentrations of 10"? to 10~4 M in water and 50 μl of a solution of herbicide (atrazine or metribuzin) in concentrations of 10"? to 10~4 M in water are added.

Atrazine-peroxidase conjugate solution (atrazine peroxidase ELIS ¹ A Kit*; Riedel de Haen) is pipetted and incubated with shaking at room temperature for 2 hours. Washing, developing and stopping are then carried out according to the instructions of the commercial assay (see above). The absorbances at 450 nm are measured in a microtiter plate reader. The results are shown in Fig. 10. No significant changes in absorbance are obtained with unmodified microtiter plates.

Table 3: Preparation of Desmetryn-embossed PP membranes (coating with BP; solutions in water; 10 min UV exposure at room temperature).

pores,
dp (μηι) Desmetryn
c(mM) Added salt
(c = 50 mM) pH Degree of functionalization
DG ^g/cm ² ) TGPl 0.2 10 1.5 863 Kl 0.2 - 1.5 724 TGP2 0.6 10 1.5 178 K2 0.6 - 1.5 132 TGP3 0.2 10 NaCl 1.5 800 K3 0.2 - NaCl 1.5 430 TGP4 0.2 10 Na3PO4 2.1 933 K4 0.2 - Na3PO4 2.1 343 TGP5 0.2 10 Na3PO4 5.1 1102 K5 0.2 - Na3PO4 5.1 559

Table 4: Desmetryn sorption from 10'* M solutions of the herbicide in water in desmetryn-imprinted polypropylene membranes and control samples (filtration rate 10 ml/min).

membrane DG ^g/cm ) Desmetrynsorption (%) PP (0.2 μια) - 48 TGP 1 (0.2 μα) 863 92 K 1 (0.2 μιη-η) 724 57 PP (0.6 μηι) - 12 TGP 2 (0.6 μα) 178 16 K 2 (0.6 μια) 132 8th

List of abbreviations

AA Acrylic acid,
AMPS 2-Acyloylamino-propane-2-sulfonic acid

BP Benzophenone

DG Degree of functionalization

EDMA ethylene glycol bismethacrylate,

FM Functional Monomer

FTIR-ATR spectroscopy - Fourier Transform Infrared - Attenuation of Total Reflection;
Infrared spectroscopy with attenuation of total reflection

K Control, synthesized without template**

Ig koW Partition coefficient 1-octano l/water

LM Solvent

MBAA N,N'-methylenebisacrylamide

s MF microfiltration

MIP Molecularly Imprinted Polymers

PhI Photoinitiator

PP Polypropylen

SEM scanning electron microscope

&iacgr;&ogr;&Tgr; Template

TGM template-embossed membranes

TGP template-imprinted polymers

UF Ultrafiltration

V Crosslinker

Claims (3)

1. Novel template-imprinted materials consisting of template-imprinted polymers (TGP) on any surface, including membranes, obtained by modifying the surface of solid supports in both organic and aqueous or aqueous-salt-containing reaction solutions, which leads to stable template imprints by way of a cross-linking polymerization of functional monomers initiated on the support surface in the presence of a template, which subsequently specifically bind template molecules or template derivatives from organic or aqueous, saline solutions. 2. Novel template-embossed materials according to claim 1, which can be produced by 1. 2.1. starting from a porous membrane, template-imprinted membranes (TGM) are synthesized while retaining the macroscopic structure and specific surface, thus obtaining both a large template binding capacity and a high permeability of the TGM, 2. 2.2. the synthesis of the template imprints is carried out by a heterogeneous photoinitiated cross-linking graft copolymerization of functional monomers on the carrier surface, 3. 2.3. a substance of the H-abstraction type is used as a photoinitiator and the membrane polymer as a coinitiator and the initiation takes place by light excitation of the photoinitiator, 4. 2.4. the synthesis of the template imprints is carried out by a chemically initiated cross-linking polymerization of functional monomers on the carrier surface, 5. 2.5. a substance is used as initiator which, after physical or chemical stimulation, generates radicals or other starter species for polymerisation, 6. 2.6. organic polymers such as polypropylene, polyethylene, polystyrene, polysulfone, polyamides, polyesters, polycarbonate, polyacrylonitrile, polyvinylidene fluoride, polytetrafluoroethylene, polyacrylates, polyacrylamides, cellulose, amylose, agarose and their respective derivatives, copolymers or blends of polymers are used as carrier materials or as membrane materials, 7. 2.7. porous solids such as glasses, silicates, ceramics or metals or their composites, also with organic polymers, are used as carrier materials or as membrane materials, 8. 2.8. Membranes with preferably symmetrical, but also asymmetrical pore structure and pore sizes between a few nm and 10 µm, preferably 100 nm to 5 µm, are used, 9. 2.9. small molecules with molecular masses below or around 100 Da, such as herbicides, active ingredients or amino acids, larger molecules such as peptides, proteins, nucleic acids or carbohydrates, or particles such as viruses, bacteria or cells, serve as templates, 10. 2.10. polymerizable compounds with groups capable of interacting with templates, in particular carboxyl, sulfonyl, sulfate, phosphate, amino or quaternary ammonium groups and their derivatives, also in mixtures, are used as functional monomers, 11. 2.11. the non-specific binding of substances competing with the template and non-templates is reduced by subsequent or prior additional functionalisation or coating. 3. Novel template-embossed materials according to claims 1 and 2, producible by 1. 3.1. crosslinking monomers and solvents for all components of the reaction mixture are also used in the mixture with the functional monomers, 2. 3.2. the addition of salt increases the binding specificity and capacity of the template-imprinted polymer for the template and template-like substances, 3. 3.3. films, membranes, fibres, hollow fibres, fabrics, fleeces or particles, each non-porous or porous, are used as carriers, 4. 3.4. the template-embossed polymer is produced without a carrier in any shape and size, 5. 3.5. the template is not removed from the template impressions after synthesis.