METHOD FOR THE DETECTION OF HORMONALLY ACTIVE COMPOUNDS, KIT AND THEIR USE

Description

[0001] The invention relates to a method for detecting hormonally active compounds by means of immobilized sulfotransferases and deformable particles as well as a kit and their use for detecting hormonally active compounds in food, detergents, dishwashing detergents, personal care products, cosmetics, pharmaceuticals, soil, water, drinking water and / or wastewater samples.

[0002] Xenohormones, or endocrine disruptors, are hormone-active compounds, specifically a heterogeneous group of compounds that affect the hormonal system of organisms. These hormone-active compounds can cause adverse health effects, including the development of breast and prostate cancer, infertility, diabetes mellitus, cardiovascular diseases, thyroid disorders, as well as neurological, neurodegenerative, and mental disorders in humans.

[0003] Due to the longevity and widespread distribution of endocrine disruptors, they are absorbed by organisms and released into the environment via a variety of routes. Firstly, endocrine disruptors can migrate from packaging into food and beverages and are thus directly absorbed through food. Systemic contamination also occurs through their dispersion in wastewater. Conventional wastewater treatment plants usually fail to adequately capture such compounds, so the contaminants enter soils, waterways, groundwater, and drinking water via sewage sludge.

[0004] Prominent representatives of xenohormones include the synthetic estrogen ethinylestradiol (EE2), the main component of hormonal contraceptives, and the "bulk chemical" bisphenol A (BPA), which is used as a raw material in the synthesis of polycarbonates and epoxy resins. The former compound is excreted predominantly in the urine in a non-metabolized form after ingestion and is considered particularly resistant to degradation. If EE2 enters the environment, for example, via sewage sludge, the consequences for aquatic habitats range from reproductive and developmental disorders in exposed organisms to the development of female sexual characteristics in male animals. According to Directive 2013 / 39 / EU of the European Parliament and of the Council, the synthetic hormone was included in the first "watch list" of pollutants potentially hazardous to the aquatic environment (Directive 2013 / 39 / EU of the European Parliament and of the Council).The measure is intended to enable EU-wide data collection to assess the risk posed by EE2.

[0005] BPA is used, among other things, in the manufacture of polycarbonate beverage bottles and for coating cans, whereby the food contained is contaminated due to direct contact with the compound. Furthermore, epoxy resin coatings on, for example, domestic plumbing pipes lead to contamination of drinking water. BPA impairs fertility and is harmful to aquatic organisms (water hazard class 2). BPA is also discussed as a possible trigger for precocious maturation and behavioral disorders. In January 2015, the European Food Safety Authority (EFSA) reduced the safe daily intake (TDI) from 50 to 4 µg / kg body weight (EFSA 2015). A legally binding limit for BPA is not currently stipulated in the Drinking Water Ordinance.However, the assessment basis for testing centers is provisional drinking water maximum values ​​derived from human toxicology for substances derived from materials that fall under the "Drinking Water Positive List Limit." Based on the TDI value and taking into account an uncertainty factor, this results in a provisional limit of 12 µg / l for drinking water.

[0006] A key prerequisite for both tracking entry routes and protecting the public from excessive exposure to xenohormones is simple and widely applicable analytical methods. While reliable methods for detecting xenohormones exist, these either fail to meet the required detection limits and thus require extensive sample preparation, or are very expensive and labor-intensive to use with laboratory diagnostic methods such as LC-MS.

[0007] The standard procedure for detecting xenohormone contamination in all types of food and drinking water is based on complex sample preparation with various extraction and purification steps and complex laboratory diagnostic detection using chromatographic methods coupled with mass spectrometry (LC-MS, GC-MS).

[0008] Furthermore, effect-related biological laboratory methods, such as the Yeast Estrogen Screen (YES) test or the Yeast Androgen Screen (YAS) test, are used to analyze hormonally active compounds, especially estrogens or androgens, in the environment. In these tests, yeast cells ( Saccharomyces cerevisiae) are used, in which the DNA sequence of the human estrogen or androgen receptor coupled to a reporter gene (e.g., lacZ encoding the enzyme β-galactosidase) has been integrated into the genome. Activation of the receptor triggers a cascade leading to the expression of the reporter gene product (e.g., β-galactosidase), which converts a substrate (e.g., chlorophenol red-β-D-galactopyranoside, CPRG). The substrate leads to a measurable color change in the medium from yellow to red (Routledge and Sumpter 1996, Sohoni and Sumpter 1998). A disadvantage of the YES and YAS tests is the use of genetically modified organisms, which is strictly regulated and therefore extremely complex in terms of laboratory technology.

[0009] Routledge and Sumpter report on the investigation of the estrogenic activity of surfactants using the YES test (Routledge and Sumpter 1996). For comparison, the natural estrogen 17β-estradiol is investigated.

[0010] The disadvantages of the YES and YAS tests are that they require extensive sample extraction and purification, respectively, and require two to three days of time. The determination of xenohormones in aqueous environmental samples is not possible with the described methods due to the low sensitivity of the yeast assay.

[0011] Bittner et al. describe the embedding of yeast cells by Saccharomyces cerevisiae into a polymer matrix, specifically gelatin and agar, to produce a yeast assay for the detection of estrogens and androgens, which can be used outside the laboratory without requiring preparation times of several days (Bittner et al. 2015). Bittner et al. describe the performance of the yeast assay for the detection of testosterone and 17β-estradiol in 3 hours using immobilized yeast cells.

[0012] Alternative systems are based on the generation of a luminescence signal (Purvis et al. 1991).

[0013] Beck et al. disclose the use of the YES test to investigate estrogens, phytoestrogens and xenoestrogens using GFP ( Green Fluorescent protein) reporter (Beck et al. 2005).

[0014] Bovee et al. describe the investigation of 17β-testosterone, 5α-dihydrotestosterone, methyltrienolone and 17β-boldenone by the YAS assay using the reporter eGFP ( enhanced Green Fluorescent Protein ) (Bovee et al. 2007).

[0015] Wolf et al. describe the investigation of androgens and anti-androgens using the YAS test in Schizosaccharomyces pompe and Saccharomyces cerevisiae using the reporter eGFP ( enhanced Green Fluorescent Protein ), thereby reducing the incubation time to 24 h compared to 48 h with the β-galactosidase reporter (Wolf et al. 2010).

[0016] WO 2014 / 106665 A1 discloses a method for detecting analytes, wherein the analyte is immobilized on hydrogel particles, which subsequently interact with a ligand immobilized on a surface. Depending on the degree of interaction, the hydrogel particles deform by adhering to the surface, allowing the deformation to be detected using reflection interference contrast microscopy.

[0017] WO 02 / 053713 A2 discloses sulfotransferases, in particular reagents and methods for regulating a human sulfotransferase. The reagents for regulating the human sulfotransferase are obtained by a process in which a test sample is contacted with the human sulfotransferase. The sulfotransferase can be present as a fusion protein and can thus be bound to a solid support.

[0018] US Patent No. 7,288,641 B1 describes variants of the sulfonetransferase 1 E1 sequence and the immobilization of SULT1E1 on a substrate. Stjernschantz et al. disclose a comparison of murine and human estrogen sulfotransferase (SULT1 E1) inhibition. in vitro and in the stone (Stjernschantz et al. 2010). Furthermore, 34 different endocrine disruptors, their inhibition of estrogen sulfotransferase, and their IC50 values ​​are described.

[0019] Du et al. disclose an immunoassay for the determination of 17β-estradiol (E 2 ) using magnetic microparticles coated with anti-E 2 antibodies as capture probes and double-codified gold nanoparticles (DC-AuNPs) modified with goat anti-rabbit antibodies and SH-ds-DNA-biotin in combination with avidin-FITC as tracer (Du et al. 2015).

[0020] Liu et al. describe a reusable fiber biosensor for the detection of 17β-estradiol (E 2 ) based on competitive binding of the sample to the human estrogen receptor (hERα) and the binding of free E 2 with fluorophore-labeled anti-E 2 antibody (Liu et al. 2017).

[0021] Cole et al. disclose the development of specific substrates for the estrogen sulfotransferase hSULT1 E1 to generate probes for the in vivo detection of the enzyme in relation to human diseases (Cole et al. 2010).

[0022] Pussak et al. describe a method for investigating interactions between molecules using specific adhesion of hydrogel particles, in particular the detection of competitive binding partners determined by the deformation of the hydrogel particle (Pussak et al. 2014).

[0023] The object of the invention is therefore to provide an easy-to-use, cost-effective and rapid method for the quantitative detection of hormonally active compounds.

[0024] The problem is solved by the features of the independent claims. Advantageous embodiments are specified in the dependent claims.

[0025] A first aspect of the invention relates to a method for detecting hormonally active compounds comprising the steps: a) Providing a surface with an immobilized analyte binding partner, wherein the analyte binding partner comprises a sulfotransferase selected from estrogen sulfotransferase hSULT1 E1 (human estrogen sulfotransferase) or a homologous estrogen sulfotransferase, b) Contacting the analyte binding partner with a sample containing the analyte, wherein the analyte is a hormonally active compound comprising at least one phenol group and interacts with the analyte binding partner, c) Contacting the analyte binding partner with a competitor, wherein the competitor is a hormonally active compound comprising at least one phenol group and is bound to a deformable particle via a functional group, wherein the deformable particle is a hydrogel particle and / or has an elastic modulus in the range of 5 kPa to 100 kPa, wherein the competitor interacts with the analyte binding partner,d) Detection of competitors bound to the analyte binding partner by detecting the deformation of the hydrogel particles.

[0026] Advantageously, the enzyme human estrogen sulfotransferase hSULT1E1 exhibits a high degree of substrate specificity similarity with the human estrogen receptor. Furthermore, the method according to the invention represents a cost-effective detection method for hormonally active compounds.

[0027] In embodiments, the method according to the invention is carried out with a sequence of steps a), b), c), and d), or a), c), b), and d), preferably with a sequence of steps a), b), c), and d). Advantageously, by contacting the analyte binding partner with the analyte before contacting with the competitor, a faster establishment of the binding equilibrium is achieved and thus a shortening of the contact times.

[0028] In an alternative embodiment, the analyte binding partner is contacted simultaneously with the analyte and the competitor.

[0029] In embodiments of the invention, the analyte binding partner is contacted with the analyte before contacting with the particle for a period of 1 min to 30 min, preferably 1 min to 20 min, particularly preferably 5 min to 15 min.

[0030] The term "immobilized" refers to a bond comprising covalent, coordinate, metallic, ionic, hydrogen bonds, and / or van der Waals interactions. Immobilization is preferably achieved through covalent or coordinate bonding.

[0031] The term "sulfotransferase" refers to an enzyme that transfers sulfonic acid groups.

[0032] The term "homologous estrogen sulfotransferase" refers to an enzyme that performs the same enzymatic reactions as the estrogen sulfotransferase hSULT1E1 and has the same substrate specificity as hSULT1E1.

[0033] In embodiments, the homologous estrogen sulfotransferase has a sequence identity of at least 85%, preferably 95%, particularly preferably 99%, with the hSULT1E1 according to SEQ ID No. 1.

[0034] In embodiments, the sulfotransferase is selected from the group comprising human sulfotransferase 1E1 (hSULT1E1), estrogen sulfotransferase isoform X1 (Pan troglodytes), Sulfotransferase family 1E estrogen-preferring member 1 (Homo sapiens), Chain A of estrogen sulfotransferase (Homo sapiens), Estrogen sulfotransferase (I place Abelius ), estrogen sulfotransferase (Nomascus leucogenys), Estrogen sulfotransferase isoform X1 (Mulatto macaque), Estrogen sulfotransferase isoform X2 (Mulatto macaque), Estrogen sulfotransferase isoform X2 (Aotus nancymaae),Estrogen sulfotransferase (Cercocebus atys), Estrogen sulfotransferase (Macaca fascicularis), Estrogen sulfotransferase (White-tailed Mandrill), Estrogen sulfotransferase (Mulatto macaque), Estrogen sulfotransferase isoform X1 (Macaca nemestrina ), estrogen sulfotransferase (Piliocolobus tephrosceles), Estrogen sulfotransferase (Theropithecus gelada), Estrogen sulfotransferase (Colobus angolensis palliatus), Estrogen sulfotransferase (Chlorocebus sabaeus), Estrogen sulfotransferase (Callithrix jacchus), Estrogen sulfotransferase (Rhinopithecus roxellana), Estrogen sulfotransferase isoform X2 ( Capuchin imitator ), estrogen sulfotransferase ( Papio Anubis ), estrogen sulfotransferase isoform X1 ( Bolivian saimiri Bolivian saimiri ), estrogen sulfotransferase isoform X1 (Aotus nancymaae), Estrogen sulfotransferase isoform X2 (Bolivian Saimiri), Estrogen sulfotransferase isoform X1 (Capuchin imitator ) and estrogen sulfotransferase ( Carlito is a syrichta ) was selected.

[0035] In preferred embodiments, the sulfotransferase is selected from the group comprising human sulfotransferase 1E1 (hSULT1E1), estrogen sulfotransferase isoform X1 (Pan troglodytes),Sulfotransferase family 1E estrogen-preferring member 1 (Homo sapiens), Chain A of estrogen sulfotransferase (Homo sapiens), Estrogen sulfotransferase (I place Abelius ), estrogen sulfotransferase (Nomascus leucogenys), Estrogen sulfotransferase isoform X1 (Mulatto macaque), Estrogen sulfotransferase isoform X2 (Mulatto macaque), Estrogen sulfotransferase isoform X2 (Aotus nancymaae), Estrogen sulfotransferase (Cercocebus atys), Estrogen sulfotransferase (Macaca fascicularis), Estrogen sulfotransferase (White-tailed Mandrill), Estrogen sulfotransferase (Mulatto macaque), Estrogen sulfotransferase isoform X1 (Macaca nemestrina ), estrogen sulfotransferase (Piliocolobus tephrosceles), Estrogen sulfotransferase (Theropithecus gelada), Estrogen sulfotransferase (Colobus angolensis palliatus ) , Estrogen sulfotransferase (Chlorocebus sabaeus), Estrogen sulfotransferase (Callithrix jacchus) and estrogen sulfotransferase (Rhinopithecus roxellana), particularly preferably from the group comprising human sulfotransferase 1E1 (hSULT1E1), estrogen sulfotransferase isoform X1 (Pan troglodytes),Sulfotransferase family 1E estrogen-preferring member 1 (Homo sapiens) and chain A of estrogen sulfotransferase (Homo sapiens); selected.

[0036] In preferred embodiments of the invention, the analyte binding partner comprises the human estrogen sulfotransferase according to SEQ ID No. 1.

[0037] In embodiments, the sample is a food, detergent, dishwashing detergent, personal care product, cosmetic, pharmaceutical, soil, water, drinking water and / or wastewater sample, preferably an aqueous solution.

[0038] The term "hormonally active compound" refers to compounds that bind to estrogen receptors, preferably estrogen receptor-α or estrogen receptor-β.

[0039] In embodiments, the analyte is a compound of formula (I) or (II) where R 1< , R 2< , R 3< , R 4< , R 6< are each independently selected from H, functional groups, unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups, and where R 5< is selected from unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups.

[0040] The term "functional group" refers to a group of atoms in a compound that determines the reactivity of the compound. In embodiments, functional groups are selected from functional groups comprising heteroatoms (O, N, S, P, or halogens), in particular carboxylic acids, halides, thiocarboxylic acids, sulfonic acids, thiol esters, sulfoxides, carboxylic anhydrides, carboxylic acid esters, carboxamides, nitriles, aldehydes, thioaldehydes, ketones, thioketones, oximes, hydrazones, alcohols, thiols, amines, imines, hydrazines, ethers, or thioethers; and functional groups without heteroatoms, in particular double bonds, triple bonds, or aromatics.

[0041] In embodiments, R 2< , R 3< , R 4< and / or R 6< is H.

[0042] In further embodiments, R 1< is an aldehyde group, an alcohol group or an esterified alcohol group.

[0043] In further embodiments, R 5< comprises a substituted ethenyl group, an aryl group or a heteroaryl group.

[0044] In embodiments, R 1< , R 2< or R 6< is a sulfate group.

[0045] In embodiments, the compound of formula (II) is bisphenol A.

[0046] In alternative embodiments, the analyte is a nonylphenol, a nonylphenol ethoxylate or a hydroxylated (poly-)halogenated aromatic hydrocarbon, in particular polychlorinated dibenzo-p-dioxins and dibenzofurans, polybrominated diphenyl ethers and halogenated bisphenol A derivatives.

[0047] The term "nonylphenol" refers to compounds of the general molecular formula C 15 H 24 O with a molecular mass of 220.35 Da, which contain a phenol group.

[0048] The term "hydroxylated (poly-)halogenated aromatic hydrocarbons" refers to halogen aromatics in which at least one hydrogen atom is replaced by a halogen, in particular fluorine, chlorine, bromine and / or iodine, and at least one hydrogen atom is replaced by a hydroxyl group.

[0049] Kester et al. disclose hydroxylated (poly-)halogenated aromatic hydrocarbons (Kester et al. 2002).

[0050] In embodiments, the analyte is selected from estrogens, preferably ethinylestradiol, estrone, estradiol, estriol, equilin or β-estradiol-17-(β-D-glucuronide); estrogen derivatives, hydroxylated (poly-)halogenated aromatic hydrocarbons, nonylphenols, bisphenol A, phenolic antiestrogens, preferably diethylstilbestrol, 4-hydroxytamoxifen, raloxifene or fulvestrant; and phenolic phytoestrogens, preferably genistein, daidzein or coumestrol.

[0051] According to the invention, the term "estrogens" refers to hormonally active compounds, particularly steroids, which have estrane as their basic structure with a phenolic A ring. Estrogens advantageously bind to the estrogen receptor.

[0052] According to the invention, the term "estrogen derivatives" refers to hormonally active compounds that have estrane with a phenolic A ring as their basic structure, as well as at least one derivatization selected from alkylation, in particular methylation; glucuronidation, halogenation, hydroxylation, methylation, and sulfation. Estrogen derivatives advantageously bind to the estrogen receptor. The term "glucuronidation" refers to the binding of a glucuronic acid.

[0053] The term "antiestrogens" refers to compounds that bind to the estrogen receptor without activation.

[0054] The term "phytoestrogens" refers to secondary plant substances that bind to the estrogen receptor and therefore have an estrogenic or anti-estrogenic effect.

[0055] In embodiments, the analyte binding partner is a fusion protein. The fusion protein comprises, for example, various protein domains with different functionalities. According to the invention, the analyte binding partner comprises a protein domain having an analyte binding site. The analyte binding site serves to bind the analyte to the analyte binding partner.

[0056] The analyte binding partner is preferably a fusion protein with a protein tag or hydrophobin, particularly preferably a fusion protein with a His-tag. The protein tag or hydrophobin advantageously enables targeted immobilization on the surface.

[0057] In embodiments, the analyte binding partner has the sequence SEQ ID No. 2.

[0058] In embodiments of the invention, the deformable particle is a functionalized particle, wherein the surface of the deformable particle has a functionalization, in particular functional groups such as carboxyl or amino groups. The functionalization serves to immobilize the competitor.

[0059] In embodiments, the deformable particle is a hydrogel particle, preferably a polyethylene glycol hydrogel particle.

[0060] In embodiments, the deformable particle has a diameter of 10 µm to 100 µm, particularly preferably a diameter of about 25 µm. The diameter of the deformable particles can be determined by bright-field optical microscopy.

[0061] According to the invention, the deformable particle has a modulus of elasticity in the range of 5 kPa to 100 kPa, preferably in the range of 10 kPa to 100 kPa, particularly preferably in the range of 15 kPa to 50 kPa. Advantageously, the modulus of elasticity in the range of 15 kPa to 50 kPa ensures high sensitivity. Furthermore, uneven deformation of the particles is advantageously excluded. The modulus of elasticity of the deformable particles can be determined from force-distance curves of atomic force spectroscopy (SFM).

[0062] In embodiments of the invention, the deformable particle has a carboxyfunctionalization. In further embodiments, the synthesis and carboxyfunctionalization of the deformable particles, in particular the hydrogel microparticles, is carried out according to the method described by Pussak et al. (Pussak et al.2012) via emulsion and radical precipitation polymerization of polyethylene glycol diacrylamide or polyethylene glycol diacrylate with subsequent radical grafting of acrylic acid monomers, crotonic acid monomers or other alkene derivatives with functional groups, such as amines, to introduce the carboxyl, amino or other functional groups.

[0063] In embodiments, the synthesis and carboxy-functionalization of the particles is carried out microfluidically by means of photoinitiated radical crosslinking of polyethylene glycol diacrylamide or polyethylene glycol diacrylate (preferred molecular weight in the range of 500 Da to 8,000 Da, particularly preferably about 4,000 Da) with subsequent photoinitiated radical grafting of acrylic acid monomers to introduce the carboxyl groups, whereby monodisperse particles with a diameter in the range of 10 µm to 100 µm, particularly preferably around 25 µm, are produced.

[0064] According to the invention, the competitor is designed to interact with the analyte binding partner, whereby the competitor and the analyte compete for interaction with the analyte binding partner. This creates competition between the free analyte in a sample and the particle-bound competitor, with an equilibrium being established depending on the analyte concentration. The more free analyte is present, the lower the binding of the particle-bound competitors to the analyte binding partner. As a result, the contact area between particle and surface decreases. Conversely, when little analyte is present in the sample to be examined, there is increased binding of the particle-bound competitors to the analyte binding partner, whereby the contact area between particle and surface increases. In embodiments of the invention, the competitor is identical to the analyte.

[0065] In embodiments, the competitor is a compound of formula (I) or (II) where R 1< , R 2< , R 3< , R 4< , R 6< are each independently selected from H, functional groups, unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups, where R 5< is selected from unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups.

[0066] In embodiments, the competitor is selected from estrogens, preferably ethinylestradiol, estrone, estradiol, estriol, equilin or β-estradiol-17-(β-D-glucuronide); estrogen derivatives, hydroxylated (poly-)halogenated aromatic hydrocarbons, nonylphenols, bisphenol A, phenolic antiestrogens, preferably diethylstilbestrol, 4-hydroxytamoxifen, raloxifene or fulvestrant; and phenolic phytoestrogens, preferably genistein, daidzein or coumestrol.

[0067] In embodiments of the invention, the competitor is selected from estrogen or an estrogen derivative, such as β-estradiol 17-(β-D-glucuronide). In preferred embodiments, the competitor is β-estradiol 17-(β-D-glucuronide).

[0068] According to the invention, the competitor is bound to a deformable particle via a functional group. In embodiments, the competitor has a functional group in position R 1< , R 2< , R 3< , R 4< , R 5< , or R 6< , preferably in position R 1< , R 2< , or R 6< , via which the competitor is bound to the deformable particle. Advantageously, the coupling in position R 1< , R 2< , or R 6< does not affect the binding of the competitor to the enzyme.

[0069] In embodiments, the competitor has a carboxyl group, a keto group, a hydroxy group, or an alkenyl, alkynyl, or aryl group, preferably a carboxyl group. The competitor is expediently coupled to the deformable particle via the carboxyl group by means of esterification or amide bonding, via the keto group by means of reductive amination, via the hydroxy group by means of esterification or condensation reaction with ether formation, via the ethynyl group by means of click chemistry, in particular copper-catalyzed 1,3-dipolar cycloaddition (Cu-catalyzed Huisgen cycloaddition), or via the alkenyl, alkynyl, or aryl group by means of a Diels-Alder reaction.

[0070] In further embodiments, the competitor is bound to the deformable particle via a linker, wherein the linker has a contour length in the range of 5 Å to 200 Å, preferably in the range of 10 Å to 65 Å; and / or a degree of polymerization in the range of 1 to 70, preferably in the range of 3 to 20; The linker is bound to the functionalized particle surface.

[0071] Advantageously, a linker molecule enables the appropriate immobilization of the competitor via the carboxyl or secondary amino group, whereby the coupling group influences the functionality and sensitivity of the method according to the invention. Furthermore, the resulting affinity of the immobilized competitor can be varied by varying the length or degree of polymerization of the linker, thus adjusting the operating range of the method according to the invention.

[0072] In embodiments, the linker is selected from the groups of homo- and heterobifunctional linkers and includes ethylenediamine, oligo- and polyethylene glycol diamines, peptides such as pentaglycine and amino acids, or a bifunctional linker with additional groups such as thiols or azides. In preferred embodiments, the linker is a polyethylene glycol diamine linker.

[0073] In some embodiments, the linkers contain protecting groups. In some embodiments of the invention, the protecting group is selected from fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, or tert-butyl protecting groups. Advantageously, the protecting groups ensure that no undesired polymerization of the linker molecules or cross-linking of the deformable particles occurs.

[0074] In embodiments of the invention, detection is carried out using quartz crystal microbalance (QCM), surface plasmon spectroscopy (SPR), atomic force spectroscopy (SFM), impedance spectroscopy, or reflection interference contrast microscopy. Preferably, detection is carried out using reflection interference contrast microscopy (RICM). The contact radii are a of particles and surface as well as the particle radii R HGS determined automatically using a specially developed software. According to the Johnson-Kendall-Roberts model (Johnson et al. 1971), these quantities are related to the adhesion energy Wadh in the following context: W adh = 4 3 a 3   E HGS / 1 − ν 2 6 π     R HGS 2 .

[0075] The determined adhesion energy corresponds to the binding density of the particle-bound competitors to the analyte binding partner and allows the determination of the amount of analyte in the sample.

[0076] In embodiments of the method according to the invention, detection is carried out using reflection interference contrast microscopy. "Reflection interference contrast microscopy" is understood to be a light microscopic method for determining layer thicknesses using interference patterns created by light reflected at the upper and lower interfaces of the layer.

[0077] In embodiments of the invention, the surface is transparent, semi-transparent, or opaque. In further embodiments, the surface is transparent at least in the range from 400 nm to 600 nm.

[0078] In embodiments of the invention, the surface is planar. For example, the surface can be a glass or plastic molded body, e.g., a microscope slide, coverslip, silicon wafer, or the like. In embodiments of the invention, the surface is designed as a well plate, in particular a 16-well or 96-well plate.

[0079] Another aspect of the invention relates to a kit comprising: i. a surface with an immobilized analyte binding partner, wherein the analyte binding partner comprises a sulfotransferase selected from estrogen sulfotransferase hSULT1E1 (human estrogen sulfotransferase) or a homologous estrogen sulfotransferase, ii. at least one deformable particle comprising an immobilized competitor, wherein the deformable particle is a hydrogel particle and / or has a modulus of elasticity in the range of 5 kPa to 100 kPa, wherein the competitor is a hormonally active compound comprising at least one phenol group and is bound to the deformable particle via a functional group, wherein the competitor interacts with the analyte binding partner.

[0080] In embodiments of the kit according to the invention, the homologous estrogen sulfotransferase has a sequence identity of at least 85%, preferably 95%, particularly preferably 99%, with the hSULT1E1 according to SEQ ID No. 1.

[0081] In embodiments of the kit according to the invention, the sulfotransferase is selected from the group comprising human sulfotransferase 1E1 (hSULT1E1), estrogen sulfotransferase isoform X1 (Pan troglodytes), Sulfotransferase family 1E estrogen-preferring member 1 (Homo sapiens), Chain A of estrogen sulfotransferase (Homo sapiens), Estrogen sulfotransferase (I place Abelius ), estrogen sulfotransferase (Nomascus leucogenys), Estrogen sulfotransferase isoform X1 (Mulatto macaque), Estrogen sulfotransferase isoform X2 (Mulatto macaque), Estrogen sulfotransferase isoform X2 (Aotus nancymaae), Estrogen sulfotransferase (Cercocebus atys), Estrogen sulfotransferase (Macaca fascicularis), Estrogen sulfotransferase (White-tailed Mandrill), Estrogen sulfotransferase (Mulatto macaque), Estrogen sulfotransferase isoform X1 (Macaca nemestrina), estrogen sulfotransferase (Piliocolobus tephrosceles), Estrogen sulfotransferase (Theropithecus gelada), Estrogen sulfotransferase (Colobus angolensis palliatus), Estrogen sulfotransferase (Chlorocebus sabaeus), Estrogen sulfotransferase (Callithrix jacchus), Estrogen sulfotransferase (Rhinopithecus roxellana), Estrogen sulfotransferase isoform X2 (Capuchin imitator ) , Estrogen sulfotransferase (Papio anubis), Estrogen sulfotransferase isoform X1 (Bolivian Saimiri), Estrogen sulfotransferase isoform X1 (Aotus nancymaae), Estrogen sulfotransferase isoform X2 (Saimiri boliviensis boliviensis), estrogen sulfotransferase isoform X1 (Capuchin imitator ) and estrogen sulfotransferase ( Carlito is a syrichta ) was selected.

[0082] In preferred embodiments, the sulfotransferase is selected from the group comprising human sulfotransferase 1E1 (hSULT1E1), estrogen sulfotransferase isoform X1 (Pan troglodytes), Sulfotransferase family 1E estrogen-preferring member 1 (Homo sapiens), Chain A of estrogen sulfotransferase (Homo sapiens), Estrogen sulfotransferase (I place Abelius), estrogen sulfotransferase (Nomascus leucogenys), Estrogen sulfotransferase isoform X1 (Mulatto macaque), Estrogen sulfotransferase isoform X2 (Mulatto macaque), Estrogen sulfotransferase isoform X2 (Aotus nancymaae), Estrogen sulfotransferase (Cercocebus atys), Estrogen sulfotransferase (Macaca fascicularis), Estrogen sulfotransferase ( Mandrillus leucophaeus), Estrogen sulfotransferase (Mulatto macaque), Estrogen sulfotransferase isoform X1 (Macaca nemestrina ), estrogen sulfotransferase (Piliocolobus tephrosceles), Estrogen sulfotransferase (Theropithecus gelada), Estrogen sulfotransferase (Colobus angolensis palliatus), Estrogen sulfotransferase (Chlorocebus sabaeus), Estrogen sulfotransferase (Callithrix jacchus) and estrogen sulfotransferase (Rhinopithecus roxellana), particularly preferably from the group comprising human sulfotransferase 1E1 (hSULT1E1), estrogen sulfotransferase isoform X1 (Pan troglodytes), Sulfotransferase family 1E estrogen-preferring member 1 (Homo sapiens) and chain A of estrogen sulfotransferase (Homo sapiens); selected.

[0083] In preferred embodiments of the kit according to the invention, the analyte binding partner comprises the human estrogen sulfotransferase according to SEQ ID No. 1.

[0084] In embodiments of the kit according to the invention, the competitor is a compound according to formula (I) or (II) where R 1< , R 2< , R 3< , R 4< , R 6< are each independently selected from H, functional groups, unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups, where R 5< is selected from unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups.

[0085] In embodiments of the kit according to the invention, the competitor is selected from estrogens, preferably ethinylestradiol, estrone, estradiol, estriol, equilin or β-estradiol-17-(β-D-glucuronide); estrogen derivatives, hydroxylated (poly-)halogenated aromatic hydrocarbons, nonylphenols, bisphenol A, phenolic antiestrogens, preferably diethylstilbestrol, 4-hydroxytamoxifen, raloxifene or fulvestrant; and phenolic phytoestrogens, preferably genistein, daidzein or coumestrol.

[0086] In embodiments of the kit according to the invention, the competitor is selected from estrogen or an estrogen derivative, such as β-estradiol 17-(β-D-glucuronide). In preferred embodiments, the competitor is β-estradiol 17-(β-D-glucuronide).

[0087] According to the invention, the competitor is bound to a deformable particle via a functional group. In embodiments of the kit according to the invention, the competitor has a functional group in position R 1< , R 2< , R 3< , R 4< , R 5< , or R 6<, preferably in position R 1< , R 2< , or R 6< , via which the competitor is bound to the deformable particle. Advantageously, the coupling in position R 1< , R 2< , or R 6< does not affect the binding of the competitor to the enzyme.

[0088] In embodiments, the competitor has a carboxyl group, a keto group, a hydroxy group, or an alkenyl, alkynyl, or aryl group, preferably a carboxyl group. The competitor is expediently coupled to the deformable particle via the carboxyl group by means of esterification or amide bonding, via the keto group by means of reductive amination, via the hydroxy group by means of esterification or condensation reaction with ether formation, via the ethynyl group by means of click chemistry, in particular copper-catalyzed 1,3-dipolar cycloaddition (Cu-catalyzed Huisgen cycloaddition), or via the alkenyl, alkynyl, or aryl group by means of a Diels-Alder reaction. In further embodiments of the kit according to the invention, the surface is transparent at least in the range from 400 nm to 600 nm.

[0089] A further aspect of the invention relates to the use of the method according to the invention and / or the kit according to the invention for the detection of hormonally active compounds in food, detergents, dishwashing detergents, personal care products, cosmetics, pharmaceuticals, soil, water, drinking water and / or wastewater samples, preferably in aqueous solutions.

[0090] To realize the invention, it is also expedient to combine the above-described embodiments and features of the claims.

[0091] The invention will be explained in more detail below using several exemplary embodiments and associated figures. These exemplary embodiments are intended to describe the invention without limiting it. The scope of the invention is defined by the claims.

[0092] The Fig. 1the schematic representation of the method according to the invention based on the competitive binding of particle-bound competitor and soluble analyte to the protein-functionalized surface. Fig. 2 the binding pockets of A) the enzyme hSULT1E1 and B) the human estrogen receptor with estradiol. Fig. 3 the adhesion energy of particles coated with estradiol-17β-D-glucuronide in interaction with hSULT1e1-coated surfaces (control), Ni-NTA surface (NiNTA), and in the presence of the analytes ethinylestradiol (EE), estrone (EST), testosterone (TE), and progesterone (PG).

[0093] The principle of the detection method is in Fig. 1The immobilized enzymes on the surface interact attractively with the particle-bound competitor, resulting in a characteristic contact area between the surface and the particle. The dissolved analyte competes with the competitor for binding sites on the surface. Depending on the analyte concentration, the contact area decreases. Above a certain concentration, the particles no longer adhere to the surface. The determination of the contact and particle radius to determine the adhesion energy is performed using reflection interference contrast microscopy. Production of a deformable particle

[0094] To generate microgel particles with defined size and shape, the droplet-based templating technique is used. The experimental setup requires two high-precision syringe pumps (one for the oil phase and one for the aqueous phase), a microfluidic flow cell with flow-focusing geometry (25 µm at the droplet-forming nozzle), a simple light microscope, and a high-speed camera for single-drop observations. Unless otherwise stated, the first dispersed phase solution contains 10% w / w PEG-diacrylamide (4-8 kDa) supplemented with 1% w / w LAP (lithium phenyl 2,4,6-trimethylbenzoylphosphinate), both dissolved in ultrapure water. The continuous phase contains 2% w / w of a surfactant (perfluoropolyether, e.g., polyhexafluoropropylene oxide, alternatively, polyacrylonitrile butadiene acrylate) dissolved in the fluorinated oil HFE 7500.After collecting the emulsions in separate Eppendorf tubes, the monomer droplets are cross-linked into microgel particles using UV light for 1 min.

[0095] To purify the hydrogel particles, 500 µl of ultrapure water is added to the collected emulsion to provide sufficient aqueous phase into which the particles can migrate. To break the emulsion and isolate the produced microspheres, 250 µl of perfluorooctanol solution (PFO, 20% (v / v) in HFE 7500) is added. The water / PFO emulsion mixture is shaken and centrifuged for 30 s at 1840 xg. The oil is removed, and another 250 µl of perfluorooctanol solution is added. The particle suspension is centrifuged once more, and the perfluorooctanol solution is removed. The resulting particles can now be functionalized using crotonic acid. Example 1: Detection of xenohormones by competitive binding

[0096] To create a functionalized surface, 24 coverslips (Menzel glasses, ø 32 mm) were placed in a Teflon rack and left in an ultrasonic bath with distilled water for 30 min. After rinsing three times with distilled water, the glasses were cleaned in denatured ethanol using an ultrasonic bath for another 30 min. The coverslips were then rinsed three times with distilled water. Further cleaning was performed using a mixture of 50 ml H 2 O 2 (35%), 50 ml of 25% aqueous NH 3 solution, and 250 ml distilled water, corresponding to a total volume of 350 ml. The solution was heated to 60°C on a hotplate, and the Teflon rack with the coverslips was left in the solution for 10 minutes. After rinsing twice with distilled water, the coverslips were dried in a nitrogen stream. To introduce amino groups, a 20 mM 3-aminopropyltriethoxysilane solution in 9:1 (vol. / vol.) isopropanol / distilled water (225 ml isopropanol, 25 ml distilled water and 1.16 ml APTES). The Teflon rack with the coverslips was then left in the solution for 120 min, thoroughly rinsed with isopropanol and dried in a nitrogen stream. The coverslips were then placed in a Petri dish and annealed at 120 °C for 60 min. For further functionalization with carboxyl groups, the coverslips were left in 250 ml of a 240 mM succinic anhydride solution (6 g in 250 ml) in tetrahydrofuran (THF) (6 g in 250 ml) for 1 h (room temperature), then washed twice with THF and once with water and dried in a nitrogen stream. To remove the chelator . N α ,N αTo couple the NTA-NH 2 -bis(carboxymethyl)lysine (NTA-NH 2 ), surfaces were first covered with 1.5 ml of a 20 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 50 mM N-hydroxysulfosuccinimide (NHS) solution in MES (3.834 mg / ml EDC and 5.75 mg / ml NHS, pH 5.0) and left at room temperature for 15 min. After the reaction solution was removed and the coverslips were washed once with MES buffer and twice with HEPES buffer (pH = 7.0), 1.5 ml of 20 mM NTA-NH 2 in HEPES was added to each coverslip and left at room temperature for 2 h. After the reaction, the surfaces were rinsed once with HEPES and once with distilled water. To remove impurities in the form of metal ions, 1.5 ml of Na 2 EDTA solution (20 mM, 7.45 mg / ml) in distilled water was added to each coverslip and left at room temperature for 30 min.The surfaces were then washed several times with distilled water, covered with 1.5 ml of a 20 mM NiCl 2 solution in citrate buffer, rinsed with distilled water after 30 min, and dried in a nitrogen stream. The citrate buffer prevents unwanted precipitation of the nickel. The Ni-NTA coating allows the immobilization of a protein with a hexahistidine sequence. For this purpose, the surfaces according to the invention were first adhered to a 16-well carrier (CS16-CultureWell™, Grace Biolabs) with a self-adhesive bottom and a volume of 400 µl / well, and the wells were washed twice with 250 µl each of loading buffer (150 mM Na 2 HPO 4 , 10 mM imidazole, pH 8.0). Subsequently, 190 µl of loading buffer was added to each well, 10 µl of a 100 µg / ml hexahistidine-SULT1e1 solution was added per well, the surfaces were incubated for 60 min at room temperature, and the surfaces were finally washed several times with loading buffer to remove nonspecifically bound protein.

[0097] To introduce amino groups, COOH-functionalized hydrogel probes with a diameter of 22.5 µm were first activated with a 20 mM EDC and 50 mM sulfoNHS solution (100 mM HEPES, pH = 7.0) under stirring. Subsequently, 50 µl of a 50 mM fluorenylmethoxycarbonyl-NH-PEG-NH 2 solution were added, and coupling was carried out over a period of 60 min. The protecting group prevents cross-linking of the particles. After the reaction, the particles were centrifuged for 10 min at 1800 x g, the supernatant was discarded, and the particles were rinsed several times with HEPES buffer (100 mM, pH = 7.0). To remove the protecting group, the particles were centrifuged again, the supernatant was discarded, and a 20% piperidine-water mixture was added. After 15 minutes of reaction, the particles were centrifuged again and rinsed several times with distilled water and HEPES buffer.For further functionalization, a 4.1 nM estradiol-17β-D-glucuronide solution (950 µl HEPES, 100 mM, pH = 7.0, 150 µl dimethyl sulfoxide) was first prepared and activated by adding EDC (18.1 mM) and s-NHS (45.4 mM) for 15 min. After activation, the estradiol derivative could be coupled to the probes by adding the mixture to the particles and allowing the reaction to proceed for two hours with stirring. The particles were then washed several times with HEPES buffer. To avoid potential interference from remaining amino groups, glycolic acid was added in a final step. For this purpose, a 50 mM glycolic acid / 20 mM EDC / 50 mM sulfo-NHS solution was prepared in HEPES buffer, the COOH group was activated for 15 min, and the mixture was then added to the hydrogel probes. After 2 hours of reaction with stirring, the particles were centrifuged and washed several times with HEPES buffer.

[0098] After the surface, for example, a glass surface, and the particles were coated, they could be used for reflection interference contrast microscopy (RICM). For this purpose, the surfaces according to the invention were adhered to a 16-well carrier (CS16-CultureWell™, Grace Biolabs) with a self-adhesive backing and a volume of 400 µl / well.

[0099] Subsequently, 200 µl of analyte solution (100 mM HEPES buffer, pH 7) was added per well. After 30 minutes of incubation of the surfaces, 10 µl of estradiol-17β-D-glucuronide-functionalized hydrogel particles were added per well, and the surfaces were examined microscopically after sedimentation of the hydrogel particles.

[0100] The radial intensity profiles of the hydrogel particles on the functionalized surface were recorded using the reflection interference contrast method using an inverted microscope system (Olympus IX 73) with a 60x immersion objective (Olympus UPlanSAPO 60x Oil Microscope Objective). Contact radii were then determined from the recorded profiles. a of particles and surface as well as the particle radii R HGS are determined automatically using specially developed software. According to the Johnson-Kendall-Roberts model, these quantities are related to the adhesion energy W adh in the following context (Johnson et al. 1971): W adh = 4 3 a 3 E HGS / 1 − ν 2 6 π R HGS 2

[0101] With a modulus of elasticity E HGS of the particles of 15 kPa and a Poisson's ratio of 0.5, the corresponding adhesion energy of the system can be determined with knowledge of the particle and contact radius.

[0102] The results of the measurements are shown as examples in Fig. 3 The estradiol-17β-D-glucuronide-functionalized particles in contact with the pure Ni-NTA surface (negative control) show only weak nonspecific interactions with the surface, whereas the probes adhere strongly to SULT1e1-coated surfaces. In the presence of high concentrations of the analyte (ethinylestradiol, estrone), the value is in the range of the negative control. This is due to the competition for SULT1e1 binding sites on the surface between free ethinylestradiol or estrone in the analyte solution and estradiol-17β-D-glucuronide bound to the hydrogel particles. Furthermore, the negligible influence of androgens, e.g., testosterone, and gestagens, e.g., progesterone, on the adhesion energy highlights the selectivity of the method towards estrogens. Cited non-patent literature

[0103] Beck V, Pfitscher A, Jungbauer A (2005) GFP-reporter for a high throughput assay to monitor estrogenic compounds. J Biochem Biophys Methods, 64(1), 19-37. Bittner M, Jarque S, Hilscherova K (2015) Polymer-immobilized ready-to-use recombinant yeast assays for the detection of endocrine disruptive compounds. Chemosphere, 132, 56-62. Bovee TFH, Helsdingen RJR, Hamers ARM, van Duursen MBM, Nielen MWF, Hoogenboom RLAP (2007) A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists. Anal Bioanal Chem, 389(5), 1549-1558. Cole GB, Keum G, Liu J, Small GW, Satayamurthy N, Keep V, Barrio JR (2010) Specific estrogen sulfotransferase (SULT1E1) Substrates and molecular imaging probe candidates Proceedings of the National Academy of Sciences 107 (14), 6222-6227. Du L, Ji W, Zhang Y, Zhang C, Liu G, Wang S (2015) An ultrasensitive detection of 17β-estradiol using a gold nano-particle-based fluorescence immunoassay. Analyst 140 (6), 2001-2007.European Food Safety Authority (EFSA) Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) (2015) Scientific Opinion on the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs: Executive summary. EFSA Journal, 13(1), 3978. Johnson KL, Kendall K, Roberts AD (1971) Surface energy and the contact of elastic solids. Proc. R. Soc. Lond. Ser. A - Math. Phys. Sci., 324, 301. Kester MHA, Bulduk S, van Toor H, Tibboel D, Meinl W, Glatt H, Falany CN, Coughtrie MWH, Schuur AG, Brouwer A, Visser TJ (2002) Potent Inhibition of Estrogen Sulfotransferase by Hydroxylated Metabolites of Polyhalogenated Aromatic Hydrocarbons Reveals Alternative Mechanism for Estrogenic Activity of Endocrine Disrupters. The Journal of Clinical Endocrinology & Metabolism, 87(3), 1142-1150. Liu L, Zhou X, Lu Y, Shan D, Xu B, He M, Shi H, Qian Y (2017) Facile screening of potential xenoestrogens by an estrogen receptor-based reusable optical biosensor.Biosensors and Bioelectronics 97, 16-20. Purvis IJ, Chotai D, Dykes CW, Lubahn DB, French FS, Wilson EM, Hobden AN (1991) An androgen-inducible expression system for Saccharomyces cerevisiae. Genes, 106, 35-42. Pussak D, Behra M, Schmidt S, Hartmann L (2012) Soft Matter, 8, 1664. Pussak D, Ponader D, Mosca S, Pompe T, Hartmann L, Schmidt S (2014) Specific Adhesion of Carbohydrate Hydrogel Particles in Competition with Multivalent Inhibitors Evaluated by AFM. Langmuir 30 (21), 6142-6150. Directive 2013 / 39 / EU of the European Parliament and of the Council (2013) DIRECTIVE 2013 / 39 / EU OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 12 August 2013 amending Directives 2000 / 60 / EC and 2008 / 105 / EC as regards priority substances in the field of water policy. Official Journal of the European Union, L 226 / 1-L 226 / 17. Routledge EJ, Sumpter JP (1996) Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen.Environmental Toxicology and Chemistry, 15 (3), 241-248. Sohoni P, Sumpter JP (1998) Several environmental oestrogens are also anti-androgens. Endocrinol, 158, 327-339. Stjernschantz E, Reinen J, Meinl W, George BJ, Glatt H, Vermeulen NPE, Oostenbrink C (2010) Comparison of murine and human estrogen sulfotransferase inhibition in vitro and in silico - Implications for differences in activity, subunit dimerization and substrate inhibition. Mol. Cell. Endocrinol. 317, 127-140. Wolf S, Rataj F, Zierau O, Ostermann K, Diel P, Parr MK, Vollmer G, Rödel G (2010) A novel combined approach to detect androgenic activities with yeast based assays in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Toxicology Letters, 199, 410-415.

Claims

1. Method for the detection of hormonally active compounds comprising the steps: a) Providing a surface with an immobilized analyte binding partner, wherein the analyte binding partner comprises a sulfotransferase selected from estrogen sulfotransferase hSULT1E1 (human estrogen sulfotransferase) or a homologous estrogen sulfotransferase, b) Contacting the analyte binding partner with a sample containing the analyte, wherein the analyte is a hormonally active compound comprising at least one phenol group and interacts with the analyte binding partner, c) Contacting the analyte binding partner with a competitor, wherein the competitor is a hormonally active compound comprising at least one phenol group and is bound to a deformable particle via a functional group, wherein the deformable particle is a hydrogel particle and / or has a Young's modulus in the range of 5 kPa to 100 kPa, wherein the competitor interacts with the analyte binding partner, d) Detection of competitors bound to the analyte binding partner by detecting the deformation of the hydrogel particles.

2. Method according to claim 1, characterized in that the homologous estrogen sulfotransferase has a sequence identity of at least 85% with hSULT1E1 according to SEQ ID No. 1.

3. Method according to claim 1 or 2, characterized in that the analyte is a compound according to formula (I) or (II) wherein R1, R2, R3, R4, R6 are each independently selected from H, functional groups, unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups, wherein R5 is selected from unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups.

4. Method according to one of claims 1 to 3 , characterized in that the analyte is selected from estrogens, estrogen derivatives, hydroxylated halogenated aromatic hydrocarbons, nonylphenols , bisphenol A, phenolic antiestrogens and phenolic phytoestrogens.

5. Method according to one of claims 1 to 4, characterized in that the analyte binding partner is a fusion protein, preferably a fusion protein with a protein tag or hydrophobin.

6. Method according to one of the preceding claims, characterized in that the competitor is a compound of formula (I) or (II) wherein R1, R2, R3, R4, R6 are each independently selected from H, functional groups, unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups, wherein R5 is selected from unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups, wherein the competitor has a functional group in position R1 , R2 or R6, via which the competitor is bound to the deformable particle.

7. Method according to one of the preceding claims, characterized in that the competitor is β-estradiol-17-(β-D-glucuronide).

8. Method according to one of the preceding claims, characterized in that the competitor is bound to the deformable particle via a linker, wherein the linker has a contour length in the range of 5 Å to 200 Å and / or a degree of polymerization in the range of 1 to 70.

9. Method according to one of the preceding claims, characterized in that the detection is carried out by means of reflection interference contrast microscopy.

10. Method according to one of the preceding claims, characterized in that the surface is transparent at least in the range of 400 nm to 600 nm.

11. Kit includes: i. a surface with an immobilized analyte binding partner, wherein the analyte binding partner comprises a sulfotransferase selected from estrogen sulfotransferase hSULT1E1 (human estrogen sulfotransferase ) or a homologous estrogen sulfotransferase, ii. at least one deformable particle comprising an immobilized competitor, wherein the deformable particle is a hydrogel particle and / or has a Young's modulus in the range of 5 kPa to 100 kPa, wherein the competitor is a hormonally active compound comprising at least one phenol group and is bound to the deformable particle via a functional group, wherein the competitor interacts with the analyte binding partner.

12. Kit according to claim 11, characterized in that the homologous estrogen sulfotransferase has a sequence identity of at least 85% with hSULT1E1 according to SEQ ID No. 1.

13. Kit according to claim 11 or 12, characterized in that the competitor is a compound according to formula (I) or (II) wherein R1, R2, R3, R4, R6 are each independently selected from H, functional groups, unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups, wherein R5 is selected from unsubstituted or substituted C1 to C15 alkyl, alkenyl or aryl groups or carbohydrate groups, wherein the competitor has a functional group in position R1 , R2 or R6, via which the competitor is bound to the deformable particle.

14. Use of a method according to one of claims 1 to 10 and / or a kit according to one of claims 11 to 13 for the detection of hormonally active compounds in food, detergent, dishwashing detergent, personal care product, cosmetic, pharmaceutical, soil, water, drinking water and / or wastewater samples.