EP1509302A1 - Feste mykotoxinträger - Google Patents

Feste mykotoxinträger

Info

Publication number
EP1509302A1
EP1509302A1 EP03755958A EP03755958A EP1509302A1 EP 1509302 A1 EP1509302 A1 EP 1509302A1 EP 03755958 A EP03755958 A EP 03755958A EP 03755958 A EP03755958 A EP 03755958A EP 1509302 A1 EP1509302 A1 EP 1509302A1
Authority
EP
European Patent Office
Prior art keywords
vinyl
methyl
acid
ethyl
mycotoxin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03755958A
Other languages
English (en)
French (fr)
Inventor
Robert Weiss
Boris Mizaikoff
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Innovationsagentur GmbH
Original Assignee
Innovationsagentur GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Innovationsagentur GmbH filed Critical Innovationsagentur GmbH
Publication of EP1509302A1 publication Critical patent/EP1509302A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/305—Addition of material, later completely removed, e.g. as result of heat treatment, leaching or washing, e.g. for forming pores
    • B01J20/3057—Use of a templating or imprinting material ; filling pores of a substrate or matrix followed by the removal of the substrate or matrix
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B01D15/3828—Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3852—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography using imprinted phases or molecular recognition

Definitions

  • the invention relates to a method for binding mycotoxins to a solid carrier.
  • DON deoxynivalenol
  • ZON zearalenone
  • the pathological pathway of DON (12, 13-Epoxy-3a, 7a, 15-trihydroxy-tri- chothec-9-en-8-one) is based on the epoxy group attached to the trichothecen ring (between C12 and C13) and the position and structure of its side groups (Binder et al . 1998).
  • ZON is chemically described as [ (-) - (3S, 11E) -3 , 4, 5, 6, 9, 10-Hexahydro-14, 16- dihydroxy-3-methyl-lH-2-benzoxa-cyclotetradecin-l, 7 (8H) -dion] and belongs to the group of resorcyclic acid lactones .
  • the standard procedures for the determination of mycotoxins involve discontinuously operated laboratory methods.
  • the common scheme for the analysis of mycotoxins is based on an extraction step followed by a time consuming clean-up procedure with a non-polar solvent and subsequent solid-phase extraction using e.g. an immunoaffinity column (Scott et al. 1993; Won-Bo et al. 1997) .
  • an immunoaffinity column e.g. an immunoaffinity column
  • extracts are then pre-concentrated and finally derivatised for the actual analysis using thin layer chromatography (TLC) , liquid chromatography (LC, HPLC) or gas- chromatographic methods such as GC/MS.
  • the method should preferably be robust (i.e. suitable for field testing without the necessity of having a laboratory facility performing such tests or methods) , specific, reliable, re-useable, and useable on an industrial scale.
  • the present invention therefore provides a method for binding mycotoxins to a solid carrier, comprising the following steps:
  • the present method provides significant advantages, especially with respect to antibodies or antibody-derived solid carriers: the storage stability of the present mycotoxin imprinted polymers exceeds 3 years, whereas antibodies are storeable for 6 month only (as a maximum) . Temperature stability is significantly enhanced with the present polymers, which makes them extremely suitable for industrial methods where high temperature steps (e.g. for pathogen de-contamination) have to be applied.
  • the present polymers are by far more resistant to (organic) solvents or against low or high pH (2-11, compared to 5 to 8 for antibodies) .
  • the present polymers have also been proven to be easily producible with surprisingly high reproducibility. Automation of the production methods for the present polymers is manageable without much effort - again, in contrast to the difficult up- scaling and automation of antibody-based techniques .
  • These developed polymers according to the present invention are suitable as solid phase extraction material for the extraction of the mycotoxin according to the present invention, especially for deoxynivalenol (DON) , 3-acetyl-deoxynivalenol (3-ac-DON) , and zearalenone (ZON) from standard solutions.
  • DON deoxynivalenol
  • 3-acetyl-deoxynivalenol 3-ac-DON
  • ZON zearalenone
  • the present polymers may also be re-used 100 times or more (depending on the specific use) for one year or even longer, whereas antibody-based methods may be used only once, at least with the initial quality criteria.
  • the present method is superior i.a. with respect to its specificity (i.e. comparable with antibody specificity), simplicity and usability on industrial scale, e.g. for food industry, beverage industry, homeland security, etc..
  • the mycotoxin imprinted polymer is specifically imprinted to a template selected from the group comprising calonectrin, deacetylcalonectrin, 7alpha, ⁇ alpha-dihydroxy- calonectrin, 7-hydroxycalonectrin, 8-hydroxy-calonectrin, 3- acetyldeoxy-nevalenol, 15-acetoxydeoxy-nivalenol, 3-acetyl-4, 7- deoxynivalen, 3, 15-diacetyl-deoxynivalenol, 4, 7-dideoxynivalenol, deoxynivalenol, fusarenon-X, nivalenol, diacetylnivalenol, 4-acetyl-scirendiol, diepoxy-diacetyl-scirpenol, 4, 15-diacetyls- cirpendiol, 3, 4-diacety
  • the mycotoxin containing solution, suspension or aerosols may be any solution, suspension or aerosol being contactable with an imprinted polymer. Even gas containing preparations with mycotoxins may be regarded as such solutions or suspensions .
  • Preferred solutions or suspensions to be contacted with the imprinted polymers according to the present invention are those common in the field of mycotoxin analytics and decontamination. Therefore, these solutions or suspensions are preferably selec- ted from the group comprising field crop preparations, food preparations, water preparations, especially waste water, rinsing water, food processing water, environmental or industrial water samples or in the atmosphere or air samples/aerosols.
  • the mycotoxin imprinted polymers are based on functional monomers selected from the group comprising 4- vinylpyridine (4-VP) , methacrylic acid (MAA) , 2-trifluoro- methylacrylic acid (TFM) , methylmethacrylates or other alkyl- methacrylates, alkylacrylates , allyl or arylacrylates and methacrylate, cyanoacrylate, styrene, alpha-methylstyrene, vinylester, especially vinylacetate, vinylchloride, methylv- inylketon, vinylidenchloride, acrylamide, methacrylamid, ac- rylonitril, methacrylonitril, 2-acetamidoacrylic acid, 2-
  • the present invention also relates to a method for preparing a mycotoxin imprinted polymer comprising the following steps:
  • Protic solvents such as water and methanol have been regarded as hindering polymerisation and disrupting the template-monomer hydrogen-bonding interactions being the prerequisite for providing a suitable imprinted polymer (Norrl ⁇ w et al, 1984) .
  • Wulff and co-workers developed polymerisable derivatives of the template molecule, which are co-polymerised with a cross-linking monomer (Wulff, 1995) . These derivatives are obtained by forming covalent bonds between the template and suitable polymerisable monomers . In order to remove the template from the polymer and liberate the binding sites, these covalent bonds have to be chemically cleaved, and are subsequently re-formed during re- binding of the target molecule.
  • Mosbach and co-workers have followed a different approach: they rely on the formation of a pre- polymerisation complex between monomers carrying suitable functional groups and the template through non-covalent bonds, such as ionic interactions or hydrogen bonding (Mosbach et al, 1996) . Following polymerisation, the functional groups are held in position by the polymeric network, whereas the template can be simply removed by solvent extraction. The principle means of re- binding the "antigen" to these polymers is again through non-covalent interactions . This self-assembly process is more similar to the natural recognition process, since most biomolecular interactions are non-covalent in nature. In summary, there are following approaches to obtain a molecularly imprinted polymer:
  • the template is bound by metal complexation to the monomer/polymer ligands like in Immobilized Metal Affinity Chromatography (IMAC) .
  • IMAC Immobilized Metal Affinity Chromatography
  • Covalent molecular imprinting The print molecule is coupled to a vinyl monomer by means of a reversible covalent linkage and the derivatised print molecule then copolymerised with an excess amount of cross-linking agent. The print molecule is then cleaved from the imprinted sites using chemical methods. Rebind- ing of the print molecules requires the original reversible bond to reform under conditions that favour substrate uptake. Most chemical linkages in the covalent approach are boronic esters, Schiff bases, and ketals. Binding sites created by covalent binding are characterised by homogeneity in binding strength throughout the whole polymer but have the major disadvantage of slow binding kinetics. In addition, it is not always possible to form covalent bonds between print molecules and functional monomers due to the lack of functionality on the print molecule.
  • Non-covalent imprinting is especially preferred according to the present invention.
  • the semi-covalent (sacrificial) molecular imprinting In this method developed by Sellergren and Andersson (Sellergren et al, 1990) the template binds covalently e.g. ester bond formation to the functional monomer during imprinting. After the imprinting process the esters are hydrolysed. During the following reaction the acid group of the functional monomer reacts to CO and the alkyl residue. The subsequent rebinding takes place by non-covalent interactions . In general the semi-covalent molecular imprinting approach should combine the homogeneity of binding sites from the covalent approach with the equilibrium of the substrate rebinding from the non-covalent approach.
  • Bio-imprinting or protein imprinting utilizes the ability of proteins to form specific binding interactions to an unnatural substrate tailored by a bioimprinting step.
  • Conventionally imprinted polymers are highly cross-linked synthetic polymers, usually linear copolymers obtained through ordinary radical polymerisation.
  • proteins consist of a primary amino acid sequence forming the ternary structure of globular proteins. Three-dimensional structures are mainly stabilised by concerted, multiple, weak interactions, including hydrogen bonds, ionic forces, and hydrophobic effects.
  • a large variety of proteins e.g. subtilisin, a protease, and bovine serum albumin (BSA) were allowed to interact with a template in aqueous solution as reported by Klibanov et al .
  • non-covalent molecular imprinting is the preferred method for providing the mycotoxin imprinted polymers according to the present invention.
  • the polymers should be rigid enough to preserve the structure of the cavity after removal of the template.
  • the polymer should be highly flexible to facilitate equilibrium between release and re-uptake of the template in the cavity.
  • accessibility of as many cavities as possible is required as well as high thermal and mechanical stability (macro porous polymers with a high inner surface area) .
  • Polymer shrinking in organic or aqueous media should be as low as possible.
  • the polymer rigidity can be crucial in the preparation of chromatographic stationary phases and surface layers e.g. for ATR crystals.
  • the print molecule or template In general, a print molecule should be soluble in organic solvents and provide suitable functional groups for interaction with the functional monomers, to ensure stable complexation.
  • the structure and chemical characteristics of the template usually determines the nature of the imprinting approach.
  • Functional monomers are generally selected for strong interactions with the template. So far there is no general protocol or rational design scheme aiding the design of molecularly imprinted polymers (MIPs) . Hence, only empirical knowledge is the starting point for the decision which ingredients should be selected.
  • MIPs molecularly imprinted polymers
  • Methacrylic acid can provide ionic interactions to basic functional groups within the template. If hydrobhobic interactions are desired, the imprint- ing solvent can be adjusted accordingly to enhance the binding strengths.
  • the most widely used functional monomer is methacrylic acid.
  • the carboxylic acid group can form hydrogen bonds and serves as proton donor, as well as a hydrogen bond acceptor.
  • methacrylic acid interacts ionically with the amine group of templates.
  • Polar functionalities like carboxylic acids, car- bamates, carboxylic esters are attracted via hydrogen bonding.
  • Methacrylic acid is suitable for templates containing Bronsted basic or hydrogen bonding functional groups close to the stereo- genie center and forms stable cyclic hydrogen bonds with templates containing acid, amide or functionalised nitrogen heterocycles .
  • MAA is broadly applicable but not a universal monomer for the generation of high affinity sites.
  • 4-Vinylpyridine (4-VP) provides better selectivity for templates containing acid groups due to its basic functionality. Additionally the p-electron system of the 4-VP benzole ring system interacts with positive charges forming ionic bonding. This property makes 4-VP an interesting candidate for MIPs suitable for applications in aqueous environments. In general, a single sort of functional monomer is used in an imprinting protocol . Nevertheless, there have been a number of approaches where combinations of two or more functional monomers, giving ter-poly- mers or higher, have yielded polymers with better recognition abilities than the recognition observed with the corresponding copolymers .
  • Cross-linking agent The cross-linking monomer is responsible for mechanical and thermal stability of the polymer. It should be able to sufficiently freeze the pre-polymerisation complex. On the other hand it should release the template after the imprinting process easily and give access to the substrate for rebinding. Hence, template leaking from the polymer is very low and the polymer backbone provides enough micro-, macro- and meso-channels for the substrate to rapidly diffuse to the binding site.
  • the cross-linking polymer is another key-factor for the selectivity due to its structure-stabilizing effect on cavities ( ⁇ 10%) . Ethylenegl col dimethacrylate (EDMA) , or tri- methylolpropane trimethacrylate (TRIM) have successfully been used.
  • Porogen/solvent The solvent plays a very important role in the generation of molecularly imprinted polymers. Three major characteristics regulate the choice of the solvent to be used: (1) it influences the structure of the MIP extensively. Besides the fact that (2) it has to dissolve template, monomer and cross-linker, (3) the solvent controls the porosity, the polymer morphology and governs the strength of non-covalent interactions.
  • Best solvents for molecular imprinting have a very low dielectric constant like toluene or chloroform (i.e. (at 20°C below 40, preferably below 20, especially below 10) .
  • MIPs imprinted with non-polar solvents mostly exhibit the best recognition for an analyte.
  • Non-polar solvents do not disturb the interaction between template and monomer in contrary to water, which is highly polar. Nevertheless, in most cases the choice of solvent is dependent on the solubility of the print molecule
  • the electrostatic interactions of the pre-polymerisation complexes are sensitive to the presence of polar protic solvents. The degree of dissociation of ion-pairs is strongly dependent on the solvent.
  • microgel particles are prepared from highly diluted monomer solutions .
  • the microgels are composed of intramolecularly crosslinked primary particles formed through multiple crosslinking.
  • the physical form of the imprinted polymers changes from phase-separated inhomogeneous blocks to macro- gel particles and finally to discrete microgel particles (Funke et al, 1998) .
  • the initiator Polymerisation is generally performed at reaction times between 16 and 48 hours, depending on the batch size and format. A number of different photo- and/or ther- molabile initiators have been used, the most common being 2,2'- azobis- (2, 4-dimethylvaleronitrile) (ABDV) and azobis- (isobutyronitrile) (AIBN) .
  • ABDV 2,2'- azobis- (2, 4-dimethylvaleronitrile
  • AIBN azobis- (isobutyronitrile)
  • the azobisnitriles are decomposed by heat (ABDV: 40°C; AIBN: 60°C) or UV light, resulting in N 2 and two metastable radicals .
  • the polymerisation process involves three principal phases: initiation, propagation, and termination. The ability of 0 2 to accept an additional electron from the radicals leads to premature chain-termination.
  • the monomer mixture is usually sparged with N 2 or He prior to polymerisation.
  • templates with antioxidant properties such as certain phenolic compounds, or molecules with long con- jugated p-electron systems, may act as scavengers to inhibit polymerisation and/or potentially be covalently incorporated in the MIP.
  • Electrostatic interactions are anticipated to be mainly responsible for generating the MIP binding sites.
  • polymerisation at low temperatures would be beneficial for the imprinting process .
  • the separation factor (a) increases with decreasing temperature of polymerisation using AIBN at 0°C.
  • Sellergren and Shea observed unchanged selectivity by thermal polymerisation. They also showed that by finishing photo initiated polymerisation with heating (120°C, 24h) higher saturation capacities and improved chromatographic performance is obtained (Sellergren et al, 1993) .
  • preferred template:monomer ratios are 1:1 to 100, especially 1:1 to 12; preferred template: crosslinker ratios are 1:1 to 100, especially 1:5 to 20. Even more preferred ratios are 1:5 to 20, especially 1:6 to 12, (template:monomer) and 1:20 to 100, especially 1:30 to 80 (template: crosslinker) .
  • Selectivity of the imprinted polymer depends on the orientation of the functional groups inside the cavities and the shape of the cavities . Selectivity increases with the number of binding interactions.
  • the template should be extracted after polymerisation under mild conditions and as complete as possible (no leaching) .
  • the equilibration with substrate molecules has to be rapid and reversible.
  • Non-co- valent imprinting based on acrylic acid requires a fourfold excess of binding sites to ensure good selectivity. Only 15% of the cavities show re-uptake of a template under these conditions; the remaining 85% are irreversibly lost for use in separation, probably because of shrinking of the cavities.
  • the design depends on the desired application of the matrix either as HPLC stationary phase (rapid adsorption/desorption: monodisperse spherical particles) or solid-phase extraction (large particles: 50mm: bulk polymerisation and subsequent grinding) .
  • HPLC stationary phase rapid adsorption/desorption: monodisperse spherical particles
  • solid-phase extraction large particles: 50mm: bulk polymerisation and subsequent grinding
  • the binding site distribution is often characterized by a small number of high affinity high selectivity binding sites and a larger class of less selective, low affinity sites. Possible solution therefore is stabilization of the template assemblies, heat treatment of the materials, and selective blocking of the low affinity non-selective sites.
  • the column efficiency is of less importance in contrast to the obtainable recovery, the column load capacity, affinity and selectivity.
  • MIPs can be prepared in various formats, the most common route is their preparation as bulk polymer with the steps of polymerisation, grinding, sieving, sedimentation and (as described hereinafter as preferred embodiment) :
  • Particle formation The polymer is mechanically ground to obtain particles with diameters in the ⁇ m range. The particles are sieved (usually 25-75 ⁇ m mesh size) and sedimented repeatedly from acetone to obtain a uniform particle size. In general, yields around 30-50% of the original bulk MIP are obtained.
  • uniform spherical polymer beads can be prepared directly by suspension polymerisation.
  • imprinted polymers particles or beads can be rendered magnetic by the inclu- sion of iron oxide, which enables facile removal of the polymer from the solvent .
  • the designed MIP has to be tested for its binding capacity of the pre-specified analyte. Usually only 15% of formed cavities can re-bind analyte molecules. Additionally the affinity for the analyte has to be determined (e.g. association constant, dissociation constant) as well as the selectivity for the analyte. There are several ways to determine these parameters . Most common is the characterisation as stationary phase in HPLC. Shift of retention time, peak asymmetry and capacity as well as separation abilities are measured. The application of MIP HPLC columns is also preferred. Molecularly imprinted binding assays using radio-labelled analyte molecules can be more easily applied with all kinds of polymers and various kinds of particle sizes. This test format preferably resembles the format of a conventional ELISA test. Spectrometric detection and immunoassays are rarely used due to sensitivity problems and the lack of availability of antibodies against the analyte, respectively.
  • Binding strength and binding-site heterogeneity are of relevance because they are responsible for the peak tailing in HPLC chromatograms making these polymers less favorable for chroma- tographic separations and/or solid-phase extraction applications .
  • MIA competitive molecularly imprinted assays
  • the present invention provides mycoctoxin MIPS . Therefore mycoc- toxins or structural analogs of mycoctoxins are used as templates.
  • Preferred templates are calonectrin, deacetylcalonectrin, 7alpha, ⁇ alpha-dihydroxy-calonectrin, 7-hy- droxycalonetrin, 8-hydroxy-calonectrin, 3-acetyldeoxy-nevalenol, 15-acetoxydeoxy-nivalenol, 3-acetyl-4, 7-deoxynivalen, 3,15-di- acetyl-deoxynivalenol, 4 , 7-dideoxynivalenol , deoxynivalenol, fusarenon-X, nivalenol, diacetylnivalenol, 4-acetyl-scirendiol, diepoxy-diacetyl-scirpenol , 4
  • Preferred functional monomers are selected from the group comprising 4-vinylpyridine (4-VP) , methacrylic acid (MAA) , 2-tri- fluoromethylacryic acid (TFM) , methylmethacrylates or other alkylmethacrylates , alkylacrylates , allyl or arylacrylates and methacrylate, cyanoacrylate, styrene, alpha-methylstyrene, vinylester, especially vinylacetate, vinylchloride, methylv- inylketon, vinylidenchloride, acrylamide, methacrylamid, ac- rylonitril, methacrylonitril, 2-acetamidoacrylic acid, 2- (acetoxyacetoxy) ethylmethacrylate 1-acetoxy-1, 3-butadiene, 2- acetoxy-3-butenenitrile, 4-acetoxystyrene, acrolein, acroleindi- ethylace
  • the porogenic solvent is preferably selected from the group comprising acetonitrile, methanol, acetone and other apolar solvents, benzene, toluene, chloroform, dichlormethane, tetrahydrofurane, dimethylformamide, dimethyl- sulfoxide, ethanol, 1-propanole, methanol, water or mixtures thereof .
  • preferred crosslinkers are selected from the group comprising di-, tri- and tetrafunctional ac- rylates or methacrylates , divinylbenzene (DVB), alkyleneglykols, polyalkyleneglycoldiacrylates und methacrylates, especially ethylenglycoldimethacrylate (EDMA) , trimethyltrimeth-acrylate (TRIM) , or ethylenglycoldiacrylate, vinyl or allylacrylates or methacrylates, diallyldiglycoldicarbonate, diallylmaleate, di- allylfumarat, diallylitaconate, vinylesters, especially divinyloxalate, divinylmalonate, diallylsuccinate, triallyliso- cyanurate, dimethacrylates or diacrylates of bis-ophenol A or ethoxylated bis-phenol A, methylene or polymethylene bisacrylam- ide or bismethayrylamide,
  • any suitable polymerisation starters may be used, however, preferably 2,2'- azobis-isobutyronitrile (AIBN), 2 , 2 ' -azobis- (2, 4-dimethylvaler- onitrile) (ABDV) or mixtures thereof are used as starters for said co-polymerisation.
  • AIBN 2,2'- azobis-isobutyronitrile
  • ABDV 2, 4-dimethylvaler- onitrile
  • the co-polymerisation is preferably carried out in an oxygen- free atmosphere.
  • the co-polymerisation is started by photoinitiation or by thermal initiation.
  • mycotoxin imprinted polymers are provided. It was surprising that with the methods according to the present invention polymers could be provided which exhibit a mycotoxin dRT (retention time difference) value of more than 0,7 thereby showing a satisfactory and specific retention of mycotoxins. Specifically, these polymers are obtainable by a method according to the present invention. With such methods, even mycotoxin dRT values of 1,0 or more, especially of 3,0 or more, are achieved. This was clearly not fo- cusable in the prior art which pointed into the opposite direction (see e.g. Jodlbauer et al . 2002), specifically for molecules having the chemical structure of DON and 2ON or similar chemical structures.
  • the preferred specific binding molecules of the polymers according to the present invention are nivalenole, deoxynivalenole, zearalenone, T2 toxin, HT2 toxin, or mixtures thereof, since these are the commercially important ones.
  • the examples of the present invention have also specifically designed in view of these preferred mycotoxins, yet without being restricted thereto.
  • the specifically preferred functional monomers, 4-vinylpyridine, methacrylic acid, 2-trifluoromethac- rylic acid or mixtures thereof, have been used for building up the polymers .
  • Specifically preferred mycotoxins to be imprinted according to the present invention include: (1) trichothecene A und B (12,13- Epoxytrichothec-9-en ring system)M especially HT-2 toxin, Di- acetoxyscirpenol, T-2 Toxin, T-2 Ttetraol, Nivalenol, Deoxynivalenol and Fusarenon-X and (2) all Zearalenone metabolites: Zearalenon, Zeranol, alpha-Zearalanol, Taleranol, beta-Zearalan- ol, alpha-Zearalenol, beta-Zearalenol .
  • the mycotoxin imprinted polymers are preferably used for solid phase extraction of mycoctoxins from mycotoxin containing solutions or suspensions, as food additives, for the preparation of a medicament for treating or preventing mycotox- in-caused disorders, for diagnosing the presence of mycotoxins in a tissue sample or fluid sample from humans, from animals or from plants .
  • the present polymers may be used in or as biosensors for detecting the presence and/or the amount of mycotoxins in a sample, especially a biological, environmental or industrial sample.
  • a preferred use on industrial scale is cleansing of mycotoxin contaminated solutions, suspensions or aerosols.
  • Food and beverage analysis and decontamination are preferred fields of use as well as water quality analysis and decontamination, e.g. if poisoned with mycotoxins (homeland security and public security application) .
  • Figure 1 Schematic of non-covalent imprinting. (1) self assembly of the print molecule (DON) , functional monomers and cross-linking monomers. After formation of the pre-polymerisation complex (2) the cross-linking monomer polymerises, (3) after extraction of the print molecule, (4) the binding site is free for specific uptake of the substrate.
  • DON print molecule
  • functional monomers functional monomers
  • cross-linking monomers polymerises
  • the binding site is free for specific uptake of the substrate.
  • FIG. 3 Chromatograms of acetone, 3-acetyl DON, fusarenon-X and DON in (a) the anti-DON MIP column DON-MAA and (b) the blank (control) polymer DON-MAA-BP.
  • Stationary phase MAA, EDMA; mobile phase: acetonitrile, flow rate 0.5 ml/min at ⁇ 8 bar backpressure, stainless steel HPLC columns (250 x 4.6 mm id); DON concentration was 1 ⁇ g/ml each.
  • Acetone concentration was 10 ⁇ l/ml each.
  • FIG. 4 Chromatograms of acetone and ZON on (a) ZON-4VP-BP, (b) ZON-4VP: anti-ZON imprinted polymer (stainless steel HPLC column: 150 x 4.6 mm id), flow rate 0.5 mL/min, mobile phase MeCN, and finally (c) QUE-4VP-BP and (d) QUE-4VP: anti-quercetin imprinted polymer (stainless steel HPLC column: 250 x 4.6 mm id), flow rate 1 ml/min, mobile phase MeCN with 0.1 % HAc. ZON concentration was 0.1 ⁇ g/ml each, acetone concentration was 10 ⁇ l/ml each.
  • Mycotoxin MIPs have numerous applications, especially in similar areas of application as conventional antibodies such as Food Analyses (Ramstr ⁇ m et al . , 2001). Due to their higher mechanical and thermal stability, MIPs can be used in applications working in harsh environments. Preferred applications of MIPs are for separation procedures in HPLC columns and solid-phase extraction (SPE) , as recognition matrix in chemical and biosensors, in molecularly imprinted sorbent assays (MIA) similar to a radio-im- muno assays (RIA) and as enzyme mimics.
  • SPE solid-phase extraction
  • MIA molecularly imprinted sorbent assays
  • MIA molecularly imprinted sorbent assays
  • RIA radio-im- muno assays
  • MIP stationary phases are used for the characterisation of MIPs.
  • Retention time is recorded for the imprinted analyte.
  • the retention time is compared to structural analogues (ideally a stereoisomer) and should be higher then the used reference.
  • the recognition properties of the polymers are then assessed by comparing the retention times (tR) or capacity factors (k') of the template with the structurally related analogues . Following parameters are generally used for the characterisation of MIPs :
  • Partition coefficient K describes the ratio of analyte distribution in the mobile phase and the stationary phase.
  • the partition coefficient K cannot be directly deduced from the chromatogram.
  • tR Total retention time tR: time required for the analytes in the mobile phase to pass through the column (at the peak maximum) .
  • tR is a characteristic signature of the analyte, but is influenced by (i) sample load and (ii) mobile phase.
  • the sample load is a frequent problem of enantiomer separation: decrease of sample loads leads to an increase in both, retention and selectivity. Furthermore the mass trans fer kinetics are particularly slow in organic mobile phases. This problem is addressed by decreasing the flow rate, (ii) When increasing the aqueous content in the mobile phases polar templates usually become less retained on MIPs, whereas templates of moderate to low polarity become more retained.
  • imprinted phases behave more like reversed phases when the aqueous content is high. This leads to pronounced specific binding, frequently in the form of total retention of all hydrophobic compounds, and can be reduced by addition of an organic modifier or a detergent.
  • Hold-up time tM time required for an analyte in the mobile phase or mobile phase molecules who do not interact with the stationary phase (not retained; also called void marker) to pass through the column (dead time) .
  • Capacity factor k' as quotient of retention time tR and hold-up time tM the capacity factor describes the enhancement of separation by the selective solid phase.
  • Preferred techniques are reversed phase, normal phase, ion ex- change, ion suppression and hydrophilic interaction chromatography (for details: see Waters Corporation, 1999)
  • MIA Molecularly Imprinted Sorbent Assay
  • the present MIPs are specifically suited as non-biological alternatives to antibodies in competitive radiolabelled molecularly imprinted sorbent assays (MIA) .
  • MIA molecularly imprinted sorbent assay
  • 3H or 14C labelled analyte is used as competitive ligand.
  • a molecularly imprinted sorbent assay (MIA) is performed similar to a RIA.
  • the bound radiolabelled analyte is detected via a radioactivity detector.
  • Binding capacity, selectivity, and affinity can be determined by assays using analogous analytes (see: US patent 6 255 461) . Selectivity in the micromolar range and lowest KD in the low nanomolar range can be observed. Heterogeneity of binding sites can be calculated by saturation studies .
  • the assay may also be designed as a competitive fluorescence assay performed with the analyte and an non related fluorescent probe and as an enzyme-linked molecularly imprinted sorbent assay using enzyme labeled substrate as competitive agent in a MIA instead of radio-labeled analytes.
  • MISPE Solid-Phase Extraction
  • SPE solid-phase extraction
  • SPE consists of percolating a known sample volume of a liquid sample through a solid sorbent (in the format of a cartridge, column or disk) under carefully selected conditions favouring the preferential absorption of the analyte over the matrix components.
  • the analyte of interest is then recovered from the sorbent by elution in a small volume (smaller than the applied sample volume) of an appropriate solvent mixture.
  • High enrichment as well as efficient sample clean up can thus be obtained in one-step.
  • Multi-purpose SPE matrices can be used for hydro- phobic analytes . The attainable enrichment and clean up depends on:
  • Common phases are hydrophobised silica (C8, C18) , styrene- divinylbenzene copolymers (PS-DVB) and graphitised carbon black (GCB) .
  • Reversed phase materials are characterized mainly by a high sample load capacity and a wide range of trapped analytes . Disadvantages are their poor selectivity, their narrow pH stability range and limited breakthrough volumes for hydrophilic analytes.
  • specificity has heretofore only be accepted to be provided by the development of high affinity SPE matrices based on biological recognition elements like enzymes, antibodies or cells.
  • MISPE the washing and elu- tion conditions need to be carefully optimised in terms of ionic strength, pH, and solvent composition. Best results are usually obtained when using the porogenic solvent as wash solvent. Possible disadvantages can be compatibility problems (many of the solvents are not miscible with water) and the drying of MIP cartridges prior to the washing step. Elution of more weakly bound analytes is generally performed by methanol or water. For more strongly bound analytes the same solvents with the addition of small amounts of acids (acetic acid, trifluoroacetic acid) or bases (TEA) are used.
  • acids acetic acid, trifluoroacetic acid
  • TAA bases
  • MIPs molecularly imprinted polymers
  • Methacrylate-based imprinted polymers have the affinity and selectivity required to act as recognition elements in a sensing device.
  • a signal is generated upon binding of the analyte to the recognition element.
  • the transducer then translates this signal into a quantifiable (one or more physicochemical parameters) output signal.
  • MIPs are used as the recognition element instead of a biomolecule.
  • Electrochemical sensors classified as amperometric, poten- tiometric, conductometric, or field effect transistor-based.
  • Mass-sensitive acoustic transducers e.g., surface-acoustic wave oscillator (SAW) , quartz crystal microbalance (QCM) .
  • SAW surface-acoustic wave oscillator
  • QCM quartz crystal microbalance
  • Optical sensors e.g., ellipsometry, surface plasmon resonance (SPR) or infrared evanescent field.
  • membranes as components of electrochemical sensors (eg. conductometric detection) .
  • an important aspect in the design of a MlP-based sensor is to find an appropriate way of interfacing the polymer with the transducer, since the MIP layer has to be in intimate contact with the transducer surface.
  • the polymer can either be synthesized in situ at the transducer surface or the surface can be coated with the preformed polymer.
  • Preferred techniques are: electro-polymerisation on conducting surfaces, spin coating and spray coating, sandwich technique, entrapping of nanometer- or micrometer-sized particles into gels or behind a membrane, spin coating of a suspension of MlP-particles in an inert, soluble polymer.
  • Methacrylate based polymers are characterized by their rigidity, which is a necessitity for HPLC solid phases, but a dissadvant- age for the formation of thin coatings . Due to the change in volume caused by solvent changes and drying, polyacrylic molecularly imprinted polymers usually crack easily on germanium (Ge) or zincselenide (ZnSe) ATR-crystals .
  • Ge germanium
  • ZnSe zincselenide
  • thin polymer layers can be produced by the sandwich technique with simple laboratory equipment, it has certain limitations. A crucial step in the sandwhich technique is the assembly of ATR crystal, the spacer (inbeneath the polymerisation liquid) and the cover glass.
  • the spacer defines the thickness of the generated polymer layer and the cover glass is necessary for the generation of an even surface and the exclusion of oxygen, which would prevent polymerization. If the polymerization is initiated by heat, the glass cover also prevents evaporation of the porogen. Otherwise the polymerization will fail, even if oxygen is excluded by polymerizing under nitrogen gas stream.
  • Ratio between functional and cross-linking monomer MAA The ratio between MAA and cross-linker can increase the "softness" and therefore the coating properties of the polymer.
  • the stability of the ATR coating is increased e.g. by changing the ratio of MAA: RIM from 1:1 to 2:1) .
  • the plasticizer dibutylphtalate was applied.
  • the polar groups of dibutylphtalate are interacting with the polar groups of the polymer. Hence, the polymer chains are loosened up and the movement of the polymer chains is facilitated. Softness and stretching of the generated polymer increases .
  • ATR-crystal materials are zincselenide or germanium, both materials have been tested as substrate for the formation of MIP layers. Adhesion of poly-acrylate based MIPs to zincselenide was more feasible, because of the high metallic surface of germanium crystals. By optimization of porogen and monomer ratio the generation of even MIP layers was realized.
  • MIPs as artificial receptors, could also be employed for the screening of libraries for potential new drugs and enzyme inhibitors.
  • the target com- pounds potential mycotoxin mimics
  • the target com- pounds can be specifically recognized by the respective MIPs from an entire library.
  • Nivalenol, fusarenon-X, 15-acetyl deoxynivalenol, 3- acetyl deoxynivalenol, quercetin, and zearalenone were supplied by Sigma-Aldrich (St. Louis, MO) .
  • the molecularly imprinted polymer (IMP) was prepared by bulk polymerisation.
  • the molar ratios of the ingredients are listed in Table 1 (DON) and Table 2
  • the polymer was then sedimented, in order to eliminate particles ⁇ 5 ⁇ m and for obtaining a uniform particle size.
  • the sedimentation was done in -250 mL acetone; after two hours the supernatant solution was discarded and fresh acetone was added to the precipitated MIP particles. The majority of small particles was eliminated after five to six sedimentations .
  • the polymer particles were filtrated, washed with methanol and dried at 45°C for 24 hours.
  • HPLC analysis The HPLC-analysis was performed using a Dionex HPLC (Dionex, Sunnyvale, CA, USA) with a P580 low pressure mixing pump and an UVD-340S diode array detector with a spectral range from 200 - 600 nm. After complete extraction of the template, each column was equilibrated with the mobile phase (MeCN with HAc) . The elution was performed at ambient temperature and monitored spectro-photometrically at 200 - 450 nm. The flow rate was kept constant at 0.5 - 1 mL min -1 throughout the whole study.
  • the aim of this example was the development and characterisation of molecularly imprinted polymers for the mycotoxins DON and ZON.
  • the major challenge for this study were the high costs for the targeted template substances.
  • only a few molecularly imprinted test polymers have been prepared after careful consideration of the imprinting procedure.
  • the isolation of the toxin from fungi cultures was pursued.
  • Both templates, DON and ZON, have OH groups as part of their molecular structure (see Figure 2) .
  • DON has three different hy- droxyl groups in 3 , 7, and 15 position of the ring system. Whereas the OH-group in position 7 is non-reactive, the other two OH-groups are reactive and are used for coupling spacers to the DON molecule.
  • the OH at position 15 is more reactive than at position 3. This reactivity is the reason for the occurrence of 3-acetyl DON and 15-acetyl DON in nature. It is therefore supposed that this two OH-groups being the most probable non-covalent interaction point for complementary functional monomers, such as methyl-methacrylate or 4-vinylpyridine.
  • the structure of ZON shows two reactive OH-groups at the main aromatic ring.
  • the two carboxyl-groups at the 14 ring are less attractive for hydrogen bonds due to their minor ability forming such bonds and, secondly, because of their less favourable accessibility due to steric hindering and due to the high flexibility of the large 14-ring system.
  • acetonitrile is one of the few solvents being able to dissolve the required amount of DON and ZON for the imprinting protocol, mainly because of its high polarity.
  • Solubility of ZON in water is only 20 mg/L; it is slightly soluble in toluol ( ⁇ 1 mg/mL) and progressively more soluble in acetonitrile (> 27 mg/mL) , methylenchloride (> 30 mg/mL), methanol, acetone and in aqueous alkali (see also Hidy et al. 1977) . Solubility of DON is even more difficult to handle; only acetonitrile (approx. 20 mg/ml) was found to be suitable (dichlormethane: 7 mg/ml; toluol: 0.4 mg/ml). Table 1 and Table 2 summarize the generated polymers for DON and ZON. Table 2 List of generated anti-ZON molecularly imprinted polymers
  • EDMA cross- linking monomer
  • deoxynivalenol shows higher retention times than the structural very similar substances 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol and fusarenon-X.
  • both control polymers DON-4VP-BP and DON- MAA-BP
  • DON-MAA-BP control polymers
  • the retention index is 1.4 times higher than in the control polymer (1.3 in DON-4VP and DON-4VP-BP) .
  • Nivalenol -2.22 12.6 0.8 17.44 1.4 0.5 0.6 0.92
  • the OH-group in position 7 is non-reactive
  • the other two OH-groups are reactive and, therefore, im- portant for the imprinting process. If these OH-groups do not exist, as it is the case in 15-acetyl and 3-acetyl DON, the recognition ability is decreased, explaining the low cross-reactivity to DON.
  • DON-4VP-BP and DON-MAA-BP nivalenol shows a higher retention time than DON most likely due to its higher hydrophobicity.
  • Imprinting of ZON was approached by conventionally using ZON in a 4-VP / EDMA co-polymer.
  • the acetone peak shifts from 2.0 min in the blank polymer (ZON-4VP-BP) to 2.2 min in the imprinted polymer (ZON-4VP)
  • the ZON peak shifts from 4.3 min to 5.4 min in the imprinted polymer (ZON-4VP and ZON-4VP-BP) .
  • the respective peak (see Figure 4 (a+b) ) is tailed, indicating an imprinting effect: in both, the blank polymer and the MIP, the same ZON concentration has been applied. Results switching to the functional monomer TFM are worse (ZON-TFM and ZON-TFM-BP) .
  • ZON has a double bond incorporated in its 15en ring system. During polymerisation this reactive double bond could be responsible for an incorporation of ZON in the subsequent polymerisation process between the functional monomer and the cross-linker. Therefore, the ZON template molecules cannot be eluted from the generated imprinted polymer, thus blocking the binding sites and resulting in a low binding capacity. Another factor adversely influencing successful imprinting can be attributed to the large ring structure. As 3-dimensional simulations of ZON show this ring system is very flexible and will not remain in a defined sterical position during polymerisation. Due to this permanent movement the formation on an exactly fitting binding site is affected during the non- covalent pre-polymerisation step. Thus created binding sites will exhibit significant structural differences, which will decrease binding strength and selectivity of the synthesized MIP matrix.
  • Quercetin is a member of the subclass of the flavonols which are secondary metabolites of various plants, contributing to sensory properties, flavour and to the texture of fruits. Electron delo- calisation throughout the whole ring system of quercetin is responsible for the antioxidant activity of quercetin and results from the formation of stable radicals (Russo et al . 2000).
  • the anti-quercetin MIP shows high retention of quercetin. Polymers were successfully imprinted against quercetin and some structural analogues like morin. Zearalenone (ZON) is less similar to quercetin than other flavone compounds, hence less selective binding and retardation in an anti-quercetin MIP is expected. With increasing acetic acid concentration, the difference between the imprinted polymer Que-4VP and the blank polymer
  • the separation factor a was determined to be 1.64 and is therefore significantly better than the previously generated ZON-4VP polymer using ZON as the template molecule.
  • DON For DON: the use of methacrylic acid and 4-Vinylpyridin as functional monomers instead of N,N-diethyl-2-aminoethylmethacrylate (DAEMA) and ethylenglykoldimethacrylate (EDMA) as crosslinker instead of divinylbenzene (DVB) .
  • DAEMA N,N-diethyl-2-aminoethylmethacrylate
  • EDMA ethylenglykoldimethacrylate
  • VB divinylbenzene
  • dummy template quercetin instead of zearalenone resulting in an increase of dRT value from 0,7 to 3,4.
  • dRT value increase was especially surprising, since up to date only the use of dummy templates from the same family of substances has been described (Matsui et al . , 2000).
  • the dummy template is a member of the flavonoide family (quercetin) , which is not related at all to the mycotoxin zearalenone.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Thermal Sciences (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
EP03755958A 2002-05-31 2003-05-30 Feste mykotoxinträger Withdrawn EP1509302A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT8372002 2002-05-31
AT8372002 2002-05-31
PCT/EP2003/005688 WO2003101580A1 (en) 2002-05-31 2003-05-30 Solid mycoctoxin carriers

Publications (1)

Publication Number Publication Date
EP1509302A1 true EP1509302A1 (de) 2005-03-02

Family

ID=29588427

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03755958A Withdrawn EP1509302A1 (de) 2002-05-31 2003-05-30 Feste mykotoxinträger

Country Status (3)

Country Link
EP (1) EP1509302A1 (de)
AU (1) AU2003242600A1 (de)
WO (1) WO2003101580A1 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111303750A (zh) * 2020-04-16 2020-06-19 湖南汇田高分子科技有限公司 水钻背面水银皮的水性保护涂料、制备方法及应用

Families Citing this family (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004036264A2 (en) 2002-10-16 2004-04-29 Georgia Tech Research Corporation Polymers, methods of use thereof, and methods of decomposition thereof
GB0509275D0 (en) * 2005-05-06 2005-06-15 Univ Cranfield Synthetic receptor
EP1988993B1 (de) 2006-02-21 2019-04-17 Mipsalus Aps Verbesserte herstellung von molekular geprägten polymeren
CN100445307C (zh) * 2006-10-09 2008-12-24 南开大学 离子液体辅助非水解溶胶-凝胶技术制备分子印迹聚合物
GB0702489D0 (en) * 2007-02-09 2007-03-21 Univ Greenwich Solid phase extraction of aflatoxins
AU2014200349B2 (en) * 2009-08-27 2015-07-23 Alltech, Inc. Synthetic mycotoxin adsorbents and methods of making and utilizing the same
CA2770343C (en) * 2009-08-27 2015-03-31 Alltech, Inc. Synthetic mycotoxin adsorbents and methods of making and utilizing the same
ES2427689B2 (es) * 2012-03-30 2014-04-07 Universidad Complutense De Madrid Materiales para el reconocimiento selectivo de micotoxinas de Alternaria (alternariol y alternariol monometil éter)
US8957164B2 (en) * 2012-06-04 2015-02-17 The Sherwin-Williams Company Coating compositions
FR3000076B1 (fr) * 2012-12-21 2015-01-16 Univ Toulon Polymere a empreinte et procede de preparation
EP2783748B1 (de) 2013-03-27 2019-05-08 Biotage AB Verwendung eines sorptionsmittels zur extraktion von mykotoxinen
CN103524676B (zh) * 2013-08-26 2014-09-17 陕西师范大学 2-[(6-氧代-6H-苯并[c]色满-3-基)氧基]丙酸整体柱的制备方法及应用
CN103936943B (zh) * 2014-04-04 2016-05-25 江苏大学 一种改性高岭土表面印迹聚合物的制备方法及其应用
DE102014008135A1 (de) * 2014-06-06 2015-12-17 Gfl Gesellschaft Für Lebensmittel-Forschung Mbh Verfahren zur analytischen Authentizitätprüfung von Inhaltsstoffen in Lebensmitteln unter Verwendung künstlicher Antikörper
CN105301147A (zh) * 2014-06-17 2016-02-03 内蒙古蒙牛乳业（集团）股份有限公司 一种液质联用法检测液态乳中己烯雌酚类激素的方法
CN104109216B (zh) * 2014-07-04 2016-05-11 山东大学 多氢键三聚氰胺核壳分子印迹聚合物及其制备方法
RU2705210C2 (ru) * 2015-02-04 2019-11-06 Олтек, Инк. Шаблоны афлатоксина, полимеры с молекулярными отпечатками и способы их получения и применения
US20160221983A1 (en) 2015-02-04 2016-08-04 Alltech, Inc. Aflatoxin templates, molecularly imprinted polymers, and methods of making and using the same
CN104931627B (zh) * 2015-05-18 2016-08-24 上海市农业科学院 一种固相萃取柱及其应用
CN105372319B (zh) * 2015-10-15 2016-08-24 济南大学 一种基于银杂化的二氧化锰纳米片构建的黄曲霉毒素b1传感器的制备方法及应用
CN105732890B (zh) * 2016-04-30 2017-07-28 中国科学院新疆理化技术研究所 一种棉酚分子印迹聚合物的制备方法
CN113008847B (zh) * 2019-07-23 2022-04-15 西南交通大学 制备喹诺酮类传感液的组合物及方法
CN111551713A (zh) * 2020-05-15 2020-08-18 中国科学院过程工程研究所 一种covid-19病毒抗体检测微球及其制备方法和含该微球的试剂盒
CN111495432B (zh) * 2020-06-05 2023-05-16 深圳职业技术学院 聚吡咯/硫化镉印迹复合光催化剂及其制备方法及应用
CN115253697B (zh) * 2021-04-29 2023-12-26 天津膜天膜科技股份有限公司 高效去除硬度纳滤膜的制备方法
CN113877552A (zh) * 2021-11-11 2022-01-04 江南大学 一种稻壳基磁性介孔二氧化硅吸附材料及其制备的固相萃取柱
CN114062576B (zh) * 2021-12-14 2023-08-25 北京普析通用仪器有限责任公司 一种分子印迹静电纺丝纤维膜及其制备方法和微流控芯片及检测玉米赤霉烯酮的方法
CN115684402B (zh) * 2022-10-25 2025-03-25 中国检验检疫科学研究院 黄曲霉毒素分离电离元件及其制备方法和试剂盒

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT370946B (de) * 1980-04-23 1983-05-10 Vana Ges M B H Mykotoxinbindendes gemisch

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03101580A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111303750A (zh) * 2020-04-16 2020-06-19 湖南汇田高分子科技有限公司 水钻背面水银皮的水性保护涂料、制备方法及应用

Also Published As

Publication number Publication date
AU2003242600A1 (en) 2003-12-19
WO2003101580A1 (en) 2003-12-11

Similar Documents

Publication Publication Date Title
WO2003101580A1 (en) Solid mycoctoxin carriers
Dirion et al. Water-compatible molecularly imprinted polymers obtained via high-throughput synthesis and experimental design
Xu et al. Molecularly imprinted solid phase extraction coupled to high-performance liquid chromatography for determination of trace dichlorvos residues in vegetables
Haupt Molecularly imprinted polymers in analytical chemistry
McNiven et al. Chloramphenicol sensor based on an in situ imprinted polymer
CN101559352B (zh) 用于检测三聚氰胺的分子印迹聚合物及其制备方法
Li et al. Selective recognition of veterinary drugs residues by artificial antibodies designed using a computational approach
Tang et al. Preparation of molecularly imprinted solid phase extraction using bensulfuron-methyl imprinted polymer and clean-up for the sulfonylurea-herbicides in soybean
Wu et al. Synthesis of bilirubin imprinted polymer thin film for the continuous detection of bilirubin in an MIP/QCM/FIA system
Andersson et al. Development of a molecularly imprinted polymer based solid-phase extraction of local anaesthetics from human plasma
Hu et al. An amobarbital molecularly imprinted microsphere for selective solid-phase extraction of phenobarbital from human urine and medicines and their determination by high-performance liquid chromatography
Feng et al. A facile molecularly imprinted polymer-based fluorometric assay for detection of histamine
Byun et al. Selective separation of aspirin using molecularly imprinted polymers
Tan et al. A new assay system for phenacetin using biomimic bulk acoustic wave sensor with a molecularly imprinted polymer coating
Ávila et al. Supported liquid membrane-modified piezoelectric flow sensor with molecularly imprinted polymer for the determination of vanillin in food samples
CA2560384C (en) Processable molecularly imprinted polymers
Zougagh et al. Automatic selective determination of caffeine in coffee and tea samples by using a supported liquid membrane-modified piezoelectric flow sensor with molecularly imprinted polymer
Allender et al. 6 Molecularly Imprinted Polymers—Preparation, Biomedical Applications and Technical Challenges
Giovannoli et al. Peptide-based affinity media for solid-phase extraction of ochratoxin A from wine samples: Effect of the solid support on binding properties
Pan et al. Synthesis and characterization of a molecularly imprinted polymer and its application as SPE enrichment sorbent for determination of trace methimazole in pig samples using HPLC-UV
Kloskowski et al. Progress in development of molecularly imprinted polymers as sorbents for sample preparation
Wang et al. Molecularly imprinted solid-phase extraction coupled with gas chromatography for the determination of four chloroacetamide herbicides in soil
Connolly et al. Evaluation of photografted charged sites within polymer monoliths in capillary columns using contactless conductivity detection
Tan et al. A piezoelectric biomimetic sensor for aminopyrine with a molecularly imprinted polymer coating
Yuan et al. Sensitive determination of rose bengal in brown sugar by a molecularly imprinted solid-phase extraction monolithic capillary column coupled with capillary electrophoresis

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20041223

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20081010

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20081202