DE102018130133A1 - Method for the detection of analytes based on immobilized proteins - Google Patents

Abstract

The invention relates to a method and a kit for the detection of analytes, in particular of low molecular weight analytes. According to the invention, a simple method for the detection of low molecular weight analytes is provided, in which the analytes to be detected act as competitors for immobilized enzymes. The inhibition allows conclusions to be drawn about the analyte concentration via the reduced substrate conversion. In particular, a method for detecting glyphosate is provided according to the invention.

Description

The invention relates to a method for the detection of analytes. In particular, the invention relates to a method for the detection of low molecular weight analytes.

The detection of low molecular weight analytes is currently complex. In particular, the detection of pesticides in drinking water and soil samples and their health effects are currently of great interest. The contamination of food including drinking water with glyphosate appears to be particularly problematic.

The detection of contaminants with pesticides in food is carried out according to the DFG multi-method S19 , with different extraction steps taking place for sample preparation, followed by analysis using LC-MS or GC-MS.

Various methods for determining glyphosate are also known in the prior art.

So the reveals CN102207495A a method for determining the glyphosate content in soil samples by means of HPLC.

Also the WO2008118899 A1 discloses a method for the detection of glyphosate by means of LC / GC coupled MS. Alternatively, detection methods using bioluminescence ( WO2010104861A1 ) and ELISA ( WO2000014538A1 ) described.

The WO2018057647 A1 describes an assay for a biochip in which pesticides can be detected on a sensor platform. The detection is based on the amplification of corresponding nucleic acids, which leads to significantly long analysis times.

The detection of corresponding low molecular weight analytes has so far been experimentally complex and not suitable to enable on-site analysis.

There is therefore a need to specify a detection method that enables simple detection of low molecular weight analytes on site.

The object of the invention is therefore to provide a method for the detection of analytes which can overcome the disadvantages in the prior art.

The object is achieved by the method according to the invention. Advantageous refinements are specified in the dependent claims.

• - Bereitstellen einer Oberfläche mit einem immobilisierten Analytbindungspartner, wobei der Analytbindungspartner eine Affinität zur Interaktion mit einem zu detektierenden Analyten aufweist,
• - Kontaktieren des Analytbindungspartners mit einer Probe enthaltend den Analyten, wobei der Analyt mit dem Analytbindungspartner interagiert,
• - Kontaktieren des Analytbindungspartners mit einem Substrat, wobei das Substrat durch den Analytbindungspartner zu einem Produkt umgesetzt wird,
• - Quantifizierung des Produkts und
• - Korrelation des quantifizierten Produkts mit einer Referenzprobe.

According to the invention, a method for the detection of analytes in a sample is proposed, comprising the steps:

• Providing a surface with an immobilized analyte binding partner, the analyte binding partner having an affinity for interaction with an analyte to be detected,
• Contacting the analyte binding partner with a sample containing the analyte, the analyte interacting with the analyte binding partner,
• Contacting the analyte binding partner with a substrate, the substrate being converted into a product by the analyte binding partner,
• - quantification of the product and
• - Correlation of the quantified product with a reference sample.

Low molecular weight analytes are understood to mean substances or molecules with a molecular weight of <800 g / mol.

In embodiments of the invention, the analyte binding partner is contacted in parallel with the analyte and the substrate.

In embodiments of the invention, the analyte binding partner is a protein or peptide having an enzymatic activity.

For the purposes of the invention, a substrate is understood to mean at least one substance which is converted by a protein domain of the analyte binding partner which has an enzymatic activity for converting the substrate to the product. In embodiments of the invention, the substrate also comprises two or more substances which are converted by the protein domain of the analyte binding partner, which has an enzymatic activity for converting the substrate to the product. In embodiments of the invention, the substrate comprises shikimate 3-phosphate and phosphoenol pyruvate.

In embodiments of the invention, the analyte binding partner has the enzymatic activity of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19). The EPSPS catalyzes the conversion of shikimate-3-phosphate and phosphoenolpyruvate to 5-enolpyruvylshikimate-3-phosphate, whereby phosphate is released. The use of the EPSPS can be used advantageously for the detection of glyphosate, since this inhibits the EPSPS and thus the detection of the presence of glyphosate in the sample to be examined is carried out via the reduced enzymatic activity. In embodiments of the invention, the analyte binding partner has the enzymatic activity of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase from E. coli (https://www.uniprot.org/uniprot/P0A6D3). The analyte binding partner preferably comprises SEQ ID No. 1.

In embodiments of the invention, the analyte binding partner is designed as a fusion protein. The fusion protein has, for example, different protein domains which have different functionalities.

In embodiments of the invention, the analyte binding partner comprises a protein domain, which has an enzymatic activity for converting the substrate to the product. The substrate can be converted to the product by the protein domain with enzymatic activity, the product or a by-product of the catalyzed reaction subsequently being detectable.

In embodiments of the invention, the analyte binding partner comprises a protein domain selected from hydrophobins, ECM proteins, S-layer proteins, peptide linkers, and protein tags. This enables directional immobilization to the surface, at least one protein domain mediating the linkage and a further protein domain having further functionality. The analyte binding partner preferably has a hydrophobin domain. In embodiments of the invention, the analyte binding partner has a hydrophobin domain and a protein domain having an enzymatic activity for converting the substrate to the product.

In embodiments of the invention, the analyte binding partner has a hydrophobin domain Ccg2 from Neurospora (N.) crassa. The analyte binding partner preferably has the SEQ. ID. No. 5 on.

In embodiments of the invention, the analyte binding partner is a fusion protein comprising a hydrophobin domain and a 5-enolpyruvylshikimate-3-phosphate synthase domain. The hydrophobin domain enables the analyte binding partner to be immobilized on the surface. In embodiments of the invention, the fusion protein has SEQ ID No. 6 on.

In embodiments of the invention, the analyte binding partner is immobilized on the surface in a mixture with hydrophobins, the mixture of the analyte binding partner with the hydrophobin in the range from 1: 2 to 1:10, preferably between 1: 3 to 1: 8, particularly preferably around 1 : 5 lies. For the purposes of the present invention, the mixing ratios given relate to the amounts of substance.

In embodiments of the invention, a mixture of the fusion protein comprising a hydrophobin domain and a 5-enolpyruvylshikimate-3-phosphate synthase domain (SEQ ID No. 6) and the hydrophobin domain Ccg2 from Neurospora (N. ) crassa (SEQ ID No. 5) in a ratio of 1: 2 to 1:10, preferably between 1: 3 to 1: 8, particularly preferably around 1: 5, very particularly preferably 1: 5.

In embodiments of the invention, the analyte binding partner is inhibited by interaction with the analyte. The analyte binding partner has an enzymatic activity which is inhibited by the interaction with the analyte. The inhibition can be competitive or non-competitive. Interaction with the analyte thus at least reduces or slows down the conversion of the substrate to the product.

In embodiments of the invention, the analyte is a crop protection agent selected from the group of the phosphonates, bipyridines, 1,3-cyclohexanediones, benzothiadiazoles, phenylpyridazines and phenoxypropionic acids.

In embodiments of the invention, the analyte is glyphosate. Glyphosate (N- (phosphonomethyl) glycine) is a non-selective leaf herbicide (broad spectrum or total herbicide) with a systemic effect that is absorbed by any green part of the plant. Glyphosate is used in agriculture against single and double germ weeds in arable, wine and fruit growing, in the cultivation of ornamental plants, on meadows, pastures and lawns as well as in the forest. The leaves absorb glyphosate by diffusion and in the plant glyphosate is distributed over the phloem. Glyphosate works by blocking the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is required for the synthesis of the aromatic amino acids phenylalanine, tryptophan and tyrosine via the shikimate route in plants, as in most microorganisms. The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is inhibited by the similarity of the glyphosate with phosphoenolpyruvate.

In embodiments of the invention, the product is quantified by optical detection. The possibility of optical detection enables simple quantification of the analyte. The optical detection is preferably carried out using a dye which allows simple optical detection.

In embodiments of the invention, the product is quantified photometrically. This enables simple quantification. The quantification is preferably carried out by correlation with a standard or a sample without analyte.

In embodiments of the invention, the product is quantified with malachite green. The phosphate content is determined using malachite green. For example, when 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is inhibited by glyphosate, the reaction with phosphoenolpyruvate is inhibited, so that conversion to 5-enolpyruvylshikimate-3-phosphate and phosphate does not take place or only slows down. The phosphate formed in the reaction can be determined by detection with malachite green and thus used as a measure for the inhibition of the EPSPS.

In embodiments of the invention, the surface on which the analyte binding partner is immobilized is formed as a particle. The surface is preferably designed as a hydrogel particle. This enables the analyte to be detected in solution. This is advantageous, for example, if a photometric determination is to be carried out.

The invention also relates to a surface having the analyte binding partner according to the invention.

In embodiments of the invention, the surface is essentially planar. For example, the surface can be a glass or plastic molded body such. B. a slide, coverslip, silicon wafer or the like. In embodiments of the invention, the surface is designed as a 96-well plate. In embodiments of the invention, the surface is designed as a particle.

In embodiments of the invention, the analyte binding partner is designed as a fusion protein. The fusion protein has, for example, different protein domains which have different functionalities.

In embodiments of the invention, the analyte binding partner comprises a protein domain, which has an enzymatic activity for converting the substrate to the product. The substrate can be converted to the product by the protein domain with enzymatic activity, the product or a by-product of the catalyzed reaction subsequently being detectable.

In embodiments of the invention, the analyte binding partner comprises a protein domain selected from hydrophobins, ECM proteins, S-layer proteins, peptide linkers, and protein tags. This enables directional immobilization to the surface, at least one protein domain mediating the linkage and a further protein domain having further functionality. The analyte binding partner preferably has a hydrophobin domain.

In embodiments of the invention, the analyte binding partner has a hydrophobin domain Ccg2 from Neurospora (N.) crassa. The analyte binding partner preferably has the SEQ. ID. No. 5 on.

In embodiments of the invention, the analyte binding partner is a fusion protein having a hydrophobin domain and at least one protein domain, having an enzymatic activity for converting the substrate to the product.

In embodiments of the invention, the analyte binding partner is a fusion protein comprising a hydrophobin domain and a 5-enolpyruvylshikimate-3-phosphate synthase domain. The hydrophobin domain enables the analyte binding partner to be immobilized on the surface. In embodiments of the invention, the fusion protein has SEQ ID No. 6 on.

In embodiments of the invention, the analyte binding partner is immobilized on the surface in a mixture with hydrophobins, the mixture of the analyte binding partner with the hydrophobin in the range from 1: 2 to 1:10, preferably between 1: 3 to 1: 8, particularly preferably around 1 : 5 lies.

In embodiments of the invention, a mixture of the fusion protein comprising a hydrophobin domain and a 5-enolpyruvylshikimate-3-phosphate synthase domain (SEQ ID No. 6) and the hydrophobin domain Ccg2 from Neurospora (N. ) crassa (SEQ ID No. 5) in a ratio of 1: 2 to 1:10, preferably between 1: 3 to 1: 8, particularly preferably around 1: 5, very particularly preferably 1: 5. By varying the ratio of fusion protein and hydrophobin on the surface, the sensitivity to glyphosate can additionally be set in a very targeted manner and thus enables detection of glyphosate over a wide concentration range.

The invention also relates to a kit for the detection of analytes comprising an analyte binding partner having an enzymatic activity for converting a substrate and a hydrophobin. The analyte binding partners and the hydrophobin contained in the kit are advantageously mixed for immobilization in a ratio of 1: 2 to 1:10, preferably 1: 3 to 1: 8, particularly preferably in a ratio of 1: 5 and immobilized on a surface. The hydrophobin stabilizes the surface while the analyte binding partner is designed to interact with the analyte.

In embodiments of the invention, the kit comprises as analyte binding partner a fusion protein comprising a hydrophobin domain and a 5-enolpyruvylshikimate-3-phosphate synthase domain.

In embodiments of the invention, the kit comprises a fusion protein with SEQ ID No. 6 and a hydrophobin with SEQ ID No. 5.

The invention also relates to the use of the method according to the invention and the kit according to the invention for the detection of analytes.

To implement the invention, it is also expedient to combine the above-described embodiments and features of the claims.

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

• 1: die schematische Darstellung des Assays, beruhend auf dem Nachweis von anorganischem Phosphat,
• 2: die Aktivitätsmessung mittels Malachitgrün-Assay bei unterschiedlichen Belegungsdichten,
• 3: die Glyphosat-abhängige Hemmung der EcEPSPS auf der Oberfläche (1 µM Ccg2_GS_EcEPSPS : 5µM Ccg2) mittels Malachitgrün-Assay,
• 4: im Vergleich dazu, die Glyphosat-abhängige Hemmung von 1µM EcEPSPS in Lösung mittels Malachitgrün-Assay,
• 5: die Entfernung verschiedener Phosphatkonzentrationen aus Lösung mittels zweier verschiedener Phosphat-bindenden Substanzen.

It shows:

• 1 : the schematic representation of the assay based on the detection of inorganic phosphate,
• 2nd : the activity measurement using a malachite green assay at different occupancy densities,
• 3rd : the glyphosate-dependent inhibition of EcEPSPS on the surface (1 µM Ccg2_GS_EcEPSPS: 5µM Ccg2) using a malachite green assay,
• 4th : in comparison, the glyphosate-dependent inhibition of 1µM EcEPSPS in solution using a malachite green assay,
• 5 : the removal of different phosphate concentrations from solution using two different phosphate-binding substances.

The principle of the detection method is in 1 shown. It is based on the malachite green assay, which is used for the general detection of inorganic phosphate ( Baykov et al. 1988 ). Phosphate must first be removed from the solution to be analyzed using a phosphate-binding substance. Then the analyte, together with the required substrates shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP), is placed on a prepared surface, for example a 96-well plate (coated with a mixture of hydrophobin Ccg2 (SEQ ID No. 4) and given the fusion protein Ccg2_GS_EcEPSPS (SEQ ID No. 6)) and incubated for 30-45 minutes. The malachite green working solution for detecting the inorganic phosphate (Pi) formed in the reaction is then added and incubated for 20 minutes in the dark. The amount of glyphosate present in the analyte solution can then be determined by measuring the absorption at 630 nm and comparing it with a control / standard.

To establish a good signal-to-noise ratio of the assay, in 2nd Different coverage densities tested on polystyrene surfaces. For this, the hydrophobin concentration was varied in particular. With increasing hydrophobin concentration, a higher enzyme function can be observed. From a hydrophobin concentration of 5 µM, no further increase in activity can be observed. The inhibition of the enzyme with 0.5 µM glyphosate can be detected at this ratio.

As proof of feasibility, in 3rd Various glyphosate concentrations were added to a functionalized glass surface (coating with 1 µM Ccg2_GS_EcEPSPS (SEQ ID No. 6): 5 µM Ccg2 (SEQ ID No. 5)) and the malachite green assay was carried out. The inhibition of enzyme activity at different glyphosate concentrations becomes clear.

Compared to 3rd , was for 4th tested the glyphosate-dependent inhibition of 1 µM EcEPSPS (SEQ ID No. 4) in solution. The inhibition of the enzyme is not visible here.

Also as proof of feasibility for 5 two different phosphate-binding substances are added to solutions with different phosphate concentrations and these are incubated for at least one hour, then centrifuged off and the remaining amount of phosphate is determined using the malachite green assay.

The malachite green assay is a well-known method for the detection of inorganic phosphate. It is based on the complexation of inorganic phosphate with molybdate. In the presence of malachite green, this complexation leads to a shift in the absorption maximum to approx. 630 nm. Accordingly, a high absorption at 630 nm indicates a high concentration of Pi. This occurs during the reaction of the EPSPS with its substrates phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P). Accordingly, the malachite green assay can be used to demonstrate the activity of the EPSPS. Glyphosate inhibits EPSPS because it competes with PEP for binding in the active enzyme center. The more Glyphosate in the surrounding medium, the lower the EPSPS activity.

In a variant of the detection method, as an alternative to a chip surface, the EcEPSPS can be coupled to suspended hydrogel microparticles in a function-preserving manner (production via emulsion and free-radical precipitation polymerization of polyethylene glycol diacrylamide with subsequent radical grafting of acrylic acid monomers to introduce the carboxyl groups, average radius 20 μm). This means that larger surfaces can be generated with immobilized EcEPSPS in smaller analyte solutions and higher sensitivities can be achieved.

Embodiment 1: Detection of glyphosate by means of a functionalized surface and malachite green assay

Fusion proteins from the hydrophobin Ccg2 from Neurospora (N.) crassa and the 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia (E.) coli (EcEPSPS) were required to create a functionalized surface. For this purpose, the coding regions of the respective genes, linked via the sequence for a flexible glycine-serine linker, were transferred into the expression vector pET28b (Novagen, Germany). The sequence of the hydrophobin (SEQ ID No. 2) is on the 5 'side and that of the EcEPSPS (SEQ ID No. 1) on the 3' side of the linker sequence. In addition, on the 5 'side of the sequence for the fusion protein there is the sequence for a (His) ₆ day, which is required for the detection and purification of the fusion protein. Furthermore, the hydrophobin without EcEPSPS was required for the surface. The gene sequence (SEQ ID No. 2) was accordingly transferred into the vector pET28b without the linker and the EcEPSPS sequence. The vectors modified in this way were, after complete sequencing of the introduced sequences, into the E. coli expression strain SHuffle T7 Express lysY (New England Biolabs, USA) transferred. This expression strain is advantageous for the expression of the hydrophobins, since it additionally codes for a disulfide bridge isomerase, which favors the correct formation of the disulfide bridges of the hydrophobins. These play an essential role in the correct folding of the protein.

Protein expression was started by adding 1 mM isopropyl-β-D-thiogalactoside (IPTG) to E. coli cells that were in the exponential growth phase. After induction, the cells were incubated for an additional 4 hours at 30 ° C and 180 revolutions per minute. The cells were then pelleted and washed so that they could then be used for protein purification. The cleaning method used depends on the solubility of the proteins. The soluble fusion proteins (Ccg2_GS_EcEPSPS, SEQ ID No. 6) were purified using nickel affinity chromatography under native conditions according to the manufacturer's instructions, while the insoluble hydrophobins were purified using denaturing nickel affinity chromatography according to the manufacturer's instructions. The hydrophobins were concentrated by ultrafiltration before dialysis; this was not necessary for the fusion proteins. After purification, the hydrophobins were reduced against a redox refolding buffer (10mM glutathione, 1mM glutathione oxidized; pH 5.4) and the fusion proteins against the Monsanto dialysis buffer (10mM MOPS, 0.5mM EDTA, 5% (v / v ) 99.9% glycerol, 1mM DTT, pH 7) dialyzed, then stored in the refrigerator and used for the functionalization of 96-well plates made of polystyrene.

The 96-well plates were functionalized by pipetting on the corresponding protein solution and then incubating for 30 minutes. The supernatant was then removed, incubated again for 5 minutes and the wells were then washed thoroughly with dialysis buffer in order to remove non-specifically bound protein. The surface was overlaid with dialysis buffer and could then be used for the malachite green assay.

The two different protein variants were required in order to be able to set an optimal ratio between the fusion protein (binds glyphosate) and hydrophobin (to stabilize the surface). For this, the protein variants were mixed in different ratios and the surfaces were functionalized. The malachite green assay was then performed. The malachite green assay is a detection method for inorganic phosphate.

By testing different ratios of fusion protein to hydrophobin it could be shown that a ratio of 1 µM fusion protein to 5 µM hydrophobin is well suited for the assay ( 2nd ). This ratio showed good EPSPS activity, ie there was a good signal-to-noise ratio. Increasing concentrations of hydrophobin brought no further increase in activity. Lower amounts of the fusion protein lead to lower signal strengths, as do higher concentrations of the fusion protein. This could be due to steric hindrance to protein activity due to too dense functionalization. Another point to be considered is that the sensitivity of the assay to glyphosate decreases with increasing fusion protein concentration, since more free binding sites have to be saturated in order to be able to measure the loss of activity in the presence of glyphosate.

The developed surface offers a decisive advantage over dissolved protein. In the concentration range used, the surface is significantly more sensitive to glyphosate than dissolved protein (comparison 3rd and 4th ). By varying the ratio of fusion protein and hydrophobin on the surface, the sensitivity to glyphosate can additionally be set in a very targeted manner and thus enables detection of glyphosate over a wide concentration range.

To prevent false positives, phosphate must be removed from the analysis solution before performing the malachite green assay. This can be achieved by using commercially available phosphate binders or by using an iron chloride solution ( 5 ).

QUOTES INCLUDE IN THE DESCRIPTION

This list of documents listed by the applicant has been generated automatically and is only included for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent literature cited

• CN 102207495 A [0005]
• WO 2008118899 A1 [0006]
• WO 2010104861 A1 [0006]
• WO 2000014538 A1 [0006]
• WO 2018057647 A1 [0007]

Non-patent literature cited

• Baykov et al. 1988 [0049]

Claims (18)

Method for the detection of analytes in a sample comprising the steps: Providing a surface with an immobilized analyte binding partner, the analyte binding partner having an affinity for interaction with an analyte to be detected, Contacting the analyte binding partner with a sample containing the analyte, the analyte interacting with the analyte binding partner, Contacting the analyte binding partner with a substrate, the substrate being converted into a product by the analyte binding partner, - quantification of the product and - Correlation of the quantified product with a reference sample. Procedure according to one of the Claims 1 , characterized in that the analyte binding partner is designed as a fusion protein. Procedure according to Claim 1 or 2nd , characterized in that the analyte binding partner comprises a protein domain, having the enzymatic activity of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Method according to one of the preceding claims, characterized in that the analyte binding partner comprises a protein domain selected from hydrophobins, preferably the hydrophobin domain Ccg2 with SEQ ID No.5 Method according to one of the preceding claims, characterized in that the analyte binding partner as a fusion protein with SEQ ID No. 6 is formed. Method according to one of the preceding claims, characterized in that the analyte binding partner is inhibited by interaction with the analyte. Method according to one of the preceding claims, characterized in that the analyte is glyphosate. Method according to one of the preceding claims, characterized in that the analyte binding partner is immobilized on the surface in a mixture with hydrophobins, the mixture of the analyte binding partner with the hydrophobin in the range from 1: 2 to 1:10, preferably between 1: 3 and 1: 8, particularly preferably around 1: 5 Procedure according to Claim 8 , characterized in that for the immobilization of the analyte binding partner a mixture of the fusion protein with the SEQ ID No. 6 and the hydrophobin domain Ccg2 with SEQ ID No. 5 in a ratio of 1: 2 to 1:10, preferably between 1: 3 to 1: 8, particularly preferably around 1: 5, very particularly preferably 1: 5. Method according to one of the preceding claims, characterized in that the quantification of the product is carried out by optical detection of the product, preferably photometrically Method according to one of the preceding claims, characterized in that the quantification of the product is carried out with malachite green. Surface having an analyte binding partner, the analyte binding partner being a fusion protein comprising a hydrophobin domain and a 5-enolpyruvylshikimate-3-phosphate synthase domain. Surface after Claim 12 , characterized in that the analyte binding partner is immobilized in a mixture with hydrophobins on the surface, the mixture of the analyte binding partner with the hydrophobin in the range from 1: 2 to 1:10, preferably between 1: 3 to 1: 8, particularly preferably around 1: 5 lies. Surface according to one of the Claims 12 or 13 , characterized in that the analyte binding partner is a fusion protein which has the SEQ ID No. 6 has. Kit for the detection of analytes comprising an analyte binding partner having an enzymatic activity for converting a substrate and a hydrophobin. Kit after Claim 15 , characterized in that the kit as analyte binding partner comprises a fusion protein having a hydrophobin domain and a 5-enolpyruvylshikimate-3-phosphate synthase domain. Kit after Claim 15 or 16 , characterized in that the kit contains a fusion protein with SEQ ID No. 6 and a hydrophobin with SEQ ID No. 5 includes. Use of a method according to one of the Claims 1 to 11 , a surface after one of the Claims 12 to 14 as well as a kit Claim 15 to 17th for the detection of analytes.

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