METHOD, SURFACE, PARTICLE AND KIT FOR THE DETECTION OF ANALYTES IN SAMPLES

Abstract

The invention relates to a method, to a surface, to a particle, and to a kit for the detection of low molecular weight analytes such as crop protection agents in samples. In particular, the invention relates to a method for the detection of glyphosate through protein-functionalised surfaces and functionalised particles by means of reflection interference contrast microscopy (RICM).

Claims

1. A method for the detection of analytes comprising the steps of: providing a surface with an immobilised analyte binding partner, contacting the analyte binding partner with a sample containing the analyte, wherein the analyte interacts with the analyte binding partner, contacting the analyte binding partner with a competitor, wherein the competitor is immobilised on a particle and interacts with the analyte binding partner, detecting the competitors bound to the analyte binding partner, wherein the analyte is glyphosate, wherein the analyte binding partner has the active centre of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) as the analyte binding site, and wherein the particle is designed to be deformable.

2. The method according to claim 1, wherein the particle is a hydrogel particle.

3. The method according to claim 1, wherein the particle has a modulus of elasticity of 5 kPa to 100 kPa.

4. The method according to claim 1, wherein the competitor is selected from phosphoenolpyruvate, phosametine, substrate analogues of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase or glyphosate.

5. The method according to claim 1, wherein the analyte binding partner is a fusion protein having a hydrophobin domain and a 5-enolpyruvylshikimate-3-phosphate synthase domain.

6. The method according to claim 1, wherein the fusion protein has SEQ ID No. 6.

7. The method according to claim 1, wherein the analyte binding partner is immobilised on the surface in a mixture with hydrophobins, the mixture of the analyte binding partner with the hydrophobin being in the range from 1:2 to 1:10.

8. The method according to claim 1, wherein the detection takes place by means of reflection interference contrast microscopy.

9. A surface having an analyte binding partner, wherein the analyte binding partner is a fusion protein having a hydrophobin domain and a 5-enolpyruvylshikimate-3-phosphate synthase domain, wherein the surface is designed to be transparent at least in the wavelength range from 400 nm to 600 nm.

10. The surface according to claim 9, wherein the analyte binding partner is immobilised on the surface in a mixture with hydrophobins, the mixture of the analyte binding partner with the hydrophobin being in the range from 1:2 to 1:10.

11. The surface according to claim 9, wherein the analyte binding partner is a fusion protein which has SEQ ID No. 6.

12. A particle having an immobilised competitor, wherein the competitor is immobilised on the particle via a linker, wherein the particle is designed to be deformable, wherein the competitor is selected from phosphoenolpyruvate, phosametine, substrate analogues of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase or glyphosate, wherein the linker has a contour length of 5-200 Å and/or a degree of polymerisation of 1-70.

13. A kit, comprising: at least one immobilised analyte binding partner or one analyte binding partner and a hydrophobin, wherein the analyte binding partner is designed to interact with an analyte and is immobilised on a surface, wherein the analyte binding partner is a fusion protein having a hydrophobin domain and a 5-enolpyruvylshikimate-3-phosphate synthase domain, and wherein the surface is designed to be transparent at least in the wavelength range from 400 nm to 600 nm; and at least one particle, having an immobilised competitor, wherein the competitor is immobilised on the particle via a linker, wherein the particle is designed to be deformable.

14. The kit according to claim 13, wherein the fusion protein has SEQ ID No. 6.

15. The kit according to claim 13, wherein the analyte binding partner is immobilised on the surface in a mixture with hydrophobins, the mixture of the analyte binding partner with the hydrophobin being in the range from 1:2 to 1:10, preferably between 1:3 to 1:8, particularly preferably around 1:5.

16. The method according to claim 1, wherein the analyte binding partner is immobilised on the surface in a mixture with hydrophobins, the mixture of the analyte binding partner with the hydrophobin being in the range from 1:3 to 1:8.

17. The method according to claim 1, wherein the analyte binding partner is immobilised on the surface in a mixture with hydrophobins, the mixture of the analyte binding partner with the hydrophobin being around 1:5.

18. The surface according to claim 9, wherein the analyte binding partner is immobilised on the surface in a mixture with hydrophobins, the mixture of the analyte binding partner with the hydrophobin being in the range from 1:3 to 1:8.

19. The surface according to claim 9, wherein the analyte binding partner is immobilised on the surface in a mixture with hydrophobins, the mixture of the analyte binding partner with the hydrophobin being around 1:5.

20. The particle according to claim 12, wherein the linker has a contour length of 10-50 Å and/or a degree of polymerisation of 3-20.

Description

EMBODIMENTS

[0119] Further features and advantages of the present invention emerge from the following schematic drawings and embodiments, on the basis of which the invention is to be explained in more detail by way of example without restricting the invention thereto.

[0120] In the drawings:

[0121] FIG. 1: is the schematic representation of the method according to the invention based on the competitive binding of particle-bound and soluble glyphosate to the protein-functionalised surface;

[0122] FIG. 2: shows the dependence of the adhesion energy between particle and surface on the concentration of glyphosate in solution and soluble glufosinate as a negative control;

[0123] FIG. 3: shows the dependence of the limit of detection and the working range of the method on the coating of the particles;

[0124] FIG. 4: shows the relative adhesion energy of the particles coated with glyphosate bound via a pentaglycine linker depending on the glyphosate concentration (soluble);

[0125] FIG. 5: shows the comparison of the relative adhesion energies of particles coated with pentaglycine glyphosate on glyphosate, other pesticides as well as glycine as a structural element of glyphosate, tested in each case at a concentration of 1 mM, except for atrazine (153 μM), chlorpyrifos (4 μM) and phosmet (79 μM).

[0126] The principle of the detection method is shown in FIG. 1. The immobilised 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 the binding sites on the surface. This reduces the contact area depending on the concentration of the analyte. Above a specific concentration, the particles do not adhere to the surface. The determination of the contact and particle radius to ascertain the adhesion energy takes place by means of reflection interference contrast microscopy.

[0127] For the detection of the specificity of the method, RCA-cleaned glass surfaces were first coated in FIG. 2 with a suitable mixture of the hydrophobin Ccg2 and the fusion protein thereof Ccg2_GS_EcEPSPS (SEQ ID No. 6). The surfaces were then incubated with either 10 mM glyphosate or glufosinate solutions and then with linker-functionalised particles with or without glyphosate coating. The adhesion energies resulting from the respective conditions were shown on the basis of a representative data set.

[0128] In FIG. 3, RCA-cleaned glass surfaces were first coated with a suitable mixture of the hydrophobin Ccg2 and the fusion protein thereof Ccg2_GS_EcEPSPS (SEQ ID No. 6). The surfaces were then incubated with glyphosate solutions of different concentrations and then with glyphosate-coated particles with ethylenediamine or pentaglycine linkers. The adhesion energies resulting from the respective conditions were shown on the basis of a representative data set.

Embodiment 1: Quantification of Glyphosate by Means of Competitive Binding

[0129] To create a functionalised surface, fusion proteins from the hydrophobin Ccg2 (SEQ ID No. 5) from Neurospora (N.) crassa and the 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia (E.) coli (EcEPSPS) (SEQ ID No. 4) were required. For this purpose, the coding regions of the respective genes, linked via the sequence for a flexible glycine-serine linker, were transferred to the expression vector pET28b (Novagen, Germany). In this case, the sequence of the hydrophobin (SEQ ID No. 2) is on the 5′ side and the sequence of the EcEPSPS (SEQ ID No. 1) is 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).sub.6 tag, 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 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 sequences introduced, transferred into the E. coli expression strain SHuffle T7 Express lysY (New England Biolabs, USA). This expression strain is advantageous for the expression of the hydrophobins, since it also codes for a disulphide bridge isomerase, which promotes the correct formation of the disulphide bridges of the hydrophobins. These play a substantial role in the correct folding of the protein.

[0130] Protein expression was started by adding 1 mM isopropyl-β-D-thiogalactoside (IPTG) to E. coli cells which were in the exponential growth phase. After induction, the cells were incubated for a further 4 hours at 30° C. and 180 revolutions per minute. The cells were then pelleted and washed so that they can then be used for protein purification. The purification method used depends on the solubility of the proteins. The soluble fusion proteins (SEQ ID No. 6) were purified by means of nickel affinity chromatography under native conditions according to the manufacturer's instructions, while the insoluble hydrophobins were purified by means of 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 dialysed against a redox refolding buffer (10 mM glutathione reduced, 1 mM glutathione oxidised; pH 5.4) and the fusion proteins were dialysed against the Monsanto dialysis buffer (10 mM MOPS, 0.5 mM EDTA, 5% (v/v) 99.9% glycerol, 1 mM DTT, pH 7), then stored in the refrigerator and used for the functionalisation of glass surfaces.

[0131] The functionalisation of glass surfaces took place by slowly pipetting on the protein solution with subsequent incubation for 30 minutes at room temperature. The surfaces were then washed thoroughly with distilled water and dried for 30 minutes at RT. Before the functionalisation, the glass surfaces were cleaned with the aid of an RCA solution (50 ml of 25% strength aqueous NH.sub.3 solution, 50 ml of 35% H.sub.2O.sub.2, 250 ml of deionised water).

[0132] The two protein variants were needed to find an optimal ratio between fusion protein (can bind glyphosate) and hydrophobin (to stabilise the surface) for the method. For this purpose, the proteins were mixed in different proportions and the functionality of the EcEPSPS on the surface was determined by means of a detection of inorganic phosphate. Inorganic phosphate is one of the reaction products in the reaction of EPSPS with its substrates phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) and can therefore be used for the detection of enzyme activity. The ratio of Ccg2 (SEQ ID No. 5) to Ccg2_GS_EcEPSPS (SEQ ID No. 6) affects the sensitivity and signal strength of the assay. The more fusion protein there is on the surface, the more glyphosate is needed to occupy the binding sites and thus measurably inhibit the activity of the proteins on the surface. Accordingly, a surface with a high concentration of fusion protein is less sensitive than one with little fusion protein. However, too low a concentration of the fusion protein results in a low signal-to-noise ratio. For this reason, various occupancy ratios were tested, resulting in a ratio of 1 μM of Ccg2_GS_EcEPSPS (SEQ ID NO. 6) to 5 μM of Ccg2 (SEQ ID No. 5) as well suited for the application.

[0133] The synthesis and carboxy functionalisation of the hydrogel microparticles took place according to the method described by Pussak et al. via emulsion and radical precipitation polymerisation of polyethylene glycol diacrylamide with subsequent radical grafting of acrylic acid monomers for the introduction of the carboxyl groups [1]. For the methods explained below, microparticles with moduli of elasticity of 15 kPa and a mean radius of 20 μm were used.

[0134] In order to allow for an interaction between the surface and the hydrogel particles or hydrogel probes (HGS) that can be modulated by the presence of the analyte, the microparticles were coated with glyphosate. For this purpose, starting from the carboxyl-functionalised HGS, various linkers were coupled to the particles. The respective linker molecule allows suitable immobilisation of the glyphosate via the carboxyl or secondary amino group, the coupling group influencing the functionality and sensitivity of the sensor. Furthermore, the resulting affinity of the immobilised competitor for the enzyme (EPSPS) can be varied via the length or the degree of polymerisation of the linker and thus the working range of the sensor can be set.

[0135] The respective coupling steps were carried out using active ester chemistry. Ethylenediamine served as a short linker variant, which was coupled to the microparticles by means of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) and 1-hydroxybenzotriazole (HOBt). For this purpose, the particles were suspended and water was replaced by dimethylformamide (DMF) in a plurality of washing steps. The particles were left in 2 ml of DMF. Subsequently, 146 mg (280 μmol) of PyBOP, 18 mg (140 μmol) of HOBt and 39 μl (280 μmol) of triethylamine were added for the activation of the carboxyl groups and the suspension was shaken at room temperature for 1 h. After adding 20 μl (300 μmol) of ethylenediamine and reacting for three hours, the particles were centrifuged at 1844×g for 10 min and in each case washed 3 times with DMF, a 1:1 mixture of DMF and water as well as pure water. For further coating, 4 mg (24 μmol) of glyphosate were dissolved in 2 ml 100 mM of Hepes buffer (pH=7.0) in an ultrasonic bath and 46 mg (240 μmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-hydrochloride (EDC) and 52 mg (240 μmol) of N-hydroxy-sulfosuccinimide sodium salt (s-NHS) were added and the carboxyl groups were activated for 15 min. The coupling of the pesticide to the amine-functionalised particles took place by combining suspension and solution and reacting over a period of 1 hour. Finally, the particles were washed 3 times with a 100 mM Hepes buffer solution (pH=7.0).

[0136] Alternatively, the particles were functionalised with a pentaglycine linker. For this purpose, the particles were washed 3 times with 2 ml of 100 mM MES buffer (pH=5.3) and the supernatant was discarded after centrifugation (10 min, 1844×g). 23 mg (120 μmol) of EDC and 26 mg (120 μmol) of s-NHS were dissolved in 1 ml of 100 mM MES buffer (pH=5.3) and the microparticles were then suspended in the solution. After activation of the carboxyl groups for one hour while shaking, 10 μl (143 μmol) of mercaptoethanol were added to inactivate the excess EDC and the suspension was left at room temperature for a further 15 min. After centrifugation (10 min, 1844×g), the supernatant was discarded and 0.2 mg (660 nmol) of the peptide dissolved in 1 ml of Hepes buffer solution (100 mM) was added, the coupling of the linker taking place overnight. After a further 3 washing steps (100 mM of Hepes buffer), the carboxyl groups were converted into the s-NHS ester according to the procedure described above, excess EDC was inactivated by means of mercaptoethanol, the suspension was centrifuged and the supernatant was discarded. After adding a 1 mg/ml of 100 mM Hepes-buffered glyphosate solution (6 μmol), the reaction mixture was left at room temperature overnight with shaking. Finally, the particles were washed 3 times with a 100 mM Hepes buffer solution (pH=7.0).

[0137] After the surface, for example a glass surface, and the particles had been coated, they could be used for reflection interference contrast microscopy (RICM). For this purpose, the surfaces according to the invention were glued to a 16-well carrier (CS16-CultureWell™, Grace Biolabs) with a self-adhesive underside and a volume of 400 μl/well.

[0138] 200 μl/well of analyte solution (100 mM HEPES buffer pH=7) were then added. After incubating the surfaces for 30 minutes, 10 μl/well of the hydrogel particles functionalised with glyphosate were added and the surfaces were microscoped after the hydrogel particles had sedimented.

[0139] The recording of the radial intensity profiles of the HGS on the functionalised surface took place in the reflection interference contrast method by means of an inverted microscope system (Olympus IX 73) with a 60× immersion objective (Olympus UPIanSAPO 60× Oil Microscope Objective). From the recorded profiles, the contact radii a of particles and surface as well as the particle radii R.sub.HES could be automatically ascertained subsequently by means of software specially developed for this purpose. According to the Johnson-Kendall-Roberts model [2], these quantities are related to the adhesion energy W.sub.adh in the following context:

[00002]$W_{\mathrm{adh}}=\frac{\frac{4}{3}{a}^3{E}_{HGS}/\left(1-v^2\right)}{6\pi R_{HGS}^2}$

[0140] With a modulus of elasticity E.sub.HGS of the particles of 15 kPa and a Poisson's number v of 0.5, the corresponding adhesion energy of the system can thus be determined with knowledge of the particle and contact radius.

[0141] The results of the measurements are shown by way of example in FIG. 2. The pentaglycine-functionalised particles (negative control) show only weak, unspecific interactions with the surface, whereas glyphosate-coated HGS adhere strongly. In the presence of high concentrations of the analyte, the value is in the range of the negative control. This is due to the competition for binding sites of the EcEPSPS on the surface between free glyphosate in the analyte solution and glyphosate bound to the HGS. Furthermore, the negligible influence of structurally similar compounds such as glufosinate on the adhesion energy illustrates the selectivity of the method compared to glyphosate.

[0142] FIG. 3 shows an example of the resulting adhesion energies of glyphosate-coated particles of the linker variants ethylenediamine and pentaglycine at different concentrations of soluble glyphosate. With increasing glyphosate concentrations in the sample, the adhesion energy of the glyphosate-coated HGS on the functionalised chip surface decreases. The more glyphosate there is in the analyte solution, the stronger the competition with bound glyphosate. If many binding sites are occupied with free glyphosate, the HGS can no longer adhere firmly to the surface, the contact area becomes smaller and the adhesion energy decreases accordingly. The limit of detection can be varied, among other things, via the linker. In the example shown, the limit of detection is 10 μM for ethylenediamine-glyphosate-coated HGS, for pentaglycine-glyphosate-coated HGS the limit of detection is below 1 μM, whereby the sensor system offers further options for setting the working range. The results show that specific detection and precise quantification of glyphosate are possible with the aid of the invention.

Embodiment 2: Determination of the Sensitivity and Specificity of the Quantification of Glyphosate

[0143] The concentration dependence of the glyphosate binding is shown in FIG. 4. For the determination of the sensitivity, RCA-cleaned glass surfaces were first coated with a suitable mixture of the hydrophobin Ccg2 and the fusion protein thereof Ccg2_GS_EcEPSPS (SEQ ID No. 6). The surfaces were then incubated with glyphosate solutions of different concentrations and then with glyphosate-coated particles with pentaglycine linkers. The examined concentration range covers a range from 10.sup.−11 M to 10.sup.−8 M. This sensitivity range reaches the threshold value of 0.1 μg/ml for pesticide contamination in German tap water.

[0144] The specificity was examined by testing structurally related compounds and frequently used pesticides in the assay according to the invention (FIG. 5). None of the tested compounds showed a relative adhesion energy comparable to that of glyphosate, whereby a specific detection of glyphosate was also proven in the presence of other pesticides and in the presence of compounds with structural elements from glyphosate.

CITED NON-PATENT LITERATURE

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