METHOD FOR THE SELECTIVE AND SENSITIVE DETECTION OF GLYPHOSATE BY PLASMONEN-ENHANCED RAMAN SPECTROSCOPY AND ITS USE

Description

Field of the invention

[0001] The present invention relates to a method for selective and sensitive detection of the herbicide glyphosate, its use to determine the presence and / or quantity, or evaluate the concentration range of glyphosate in a sample, and a kit to carry out the mentioned method.State of the art

[0002] Environmental pollution, protection and restoration. Clean vs. contaminated water, soil and air and its direct impact on human health and life, on its quality. Specific official guidelines and regulations of governments and other relevant legal institutions are presented more frequently and forcefully on the local, regional and global levels whose observance is exceedingly important for environmental protection, and consequently, for the protection of human health and life. Currently, there is a wide range of chemical substances and products, now also legally defined, the use of which is prohibited or at least significantly restricted.

[0003] In general, regulatory and control procedures for determining the concentration levels of various pollutants require long monitoring times, usually using traditional methods: sampling, sending to certified laboratories, and time-consuming and costly analyzes (mostly using gas / liquid chromatography and / or mass spectrometry). However, the results obtained in this way are not always unambiguous, either due to the time-consuming nature of the analyses or to impossibility of avoiding many demanding preparatory procedures.

[0004] The problem is also that many of the molecules - important from the point of view of environmental and human health protection possess characteristics that naturally obstacle their selective and sensitive detection. Even more, their detection itself is often a big challenge. It is also the case of glyphosate, a broad-spectrum herbicide used even today relatively commonly and often against weeds (e.g. Roundup product which is also sold and used in the Slovak Republic). During the 20th century, the use of glyphosate increased enormously, mainly due to genetically modified crops and the subsequent higher resistance of plants to its action. This resulted in the gradual accumulation of glyphosate in the environment (soil, water, etc.) as well as in food ingredients and products. The use of glyphosate is not banned currently, but its harmfulness - probable carcinogenicity - is often discussed and the issue of its monitoring is becoming increasingly important from the point of view of the EU and its environmental policy (current European legislation allows use of glyphosate until December 15, 2023).

[0005] Raman spectroscopy (RS), especially plasmon-enhanced Raman spectroscopy (PERS), has proven to be a suitable analytical technique for several decades, which provides monitoring or 'on site' quick screening of samples without the need for the (laborious) sample pre-treatment, and which is capable of detecting and distinguishing individual pollutants even in various mixtures and at low concentrations. In addition, it is shown to be a technique that is capable of detecting a wide range of chemicals in the desired ppb-ppm concentration range. However, direct detection of glyphosate using Raman and PERS spectroscopy is significantly limited since these spectra can only be detected at high concentrations of the herbicide. Therefore, there is a need for chemical modification of the glyphosate molecule which can result in a compound - final product - with a characteristic PERS spectrum by which a selective and sensitive detection of glyphosate in an aqueous environment even at low concentrations of glyphosate can be carried out.

[0006] The paper from DE GOES RAFAEL ELEODORO ET AL: "Tuning of Citrate-Stabilized Laser Ablated Silver Nanoparticles for Glyphosate Detection",IEEE SENSORS JOURNAL, IEEE, USA, vol. 20, no. 4, 28 October 2019, pages 1843-1850,discloses a method of detection of glyphosate in water. Aqueous glyphosate samples are prepared employing deionized water and then combined with silver nanoparticles in a colloidal solution. The resulting solution is interrogated with a Surface Enhanced Raman Scattering spectrometer in order to determine the glyphosate concentration. This document fails to discloses in particular the mixing of the aqueous solution of the sample containing glyphosate with carbon disulfide in an alkaline environment. The paper from BHATT V K ET AL: "A new spectrophotometric method for the determination of glyphosate: statistical optimization and application in biodegradation studies",INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCE AND TECHNOLOGY, vol. 18, no. 4, 15 February 2020, pages 997-1008, discloses a spectrophotometric method for the determination of glyphosate where a sample containing glyphosate has being reacted with carbon disulfide and copper(II)nitrate in alkaline medium. The yellow colored aqueous phase obtained by this mixing is then investigated by UV-absorption spectroscopy. This method is however not concerned with enhanced Raman spectroscopy.

[0007] The shortcomings of the closest prior art mentioned above are solved by the present invention.Summary of the invention

[0008] The subject of the present invention is a method of detecting glyphosate using plasmon-enhanced Raman spectroscopy (PERS) which is based on a suitable chemical modification of the glyphosate molecule in order to change its chemical structure, so that the modified molecule contains functional groups demonstrating a significantly higher affinity towards the nanostructured (nanoparticle) plasmonic surfaces. In this way, the conditions for the possible detection of glyphosate found in the samples in an aqueous environment using the plasmon-enhanced Raman signal (PERS) will be achieved even at (very) low concentrations of the given herbicide. Taking into account that the PERS detection of the selected environmental pollutants is selective, sensitive, time- and cost-effective technique, thus, it provides an accessible screening method suitable for monitoring the presence of glyphosate in an aqueous environment together with possible quantification of the molecule, and therefore, possible verification of the thresholds of the presence of detected molecules.

[0009] The benefit of the present invention is the use of relatively simple technical equipment, inexpensive reagents, time-efficient assessment and the possibility to perform 'on site' analysis at the place of the sample collection, assuming a portable Raman spectrometer is employed.

[0010] The subject of the present invention is a method for detection of the herbicide glyphosate using plasmon-enhanced Raman spectroscopy (PERS) which is catalytically mediated by silver plasmonic nanoparticles.

[0011] An appropriate chemical modification of the glyphosate molecule in the sense of the present invention is represented by the chemical reaction of an aqueous solution of a sample containing glyphosate with carbon disulfide in an alkaline environment which results in a carbon disulfide-modified glyphosate, which in an acidic environment demonstrates an affinity towards the silver surface in the form of nanoparticles, which is significantly higher than that of the glyphosate molecules, which enables its adsorption on and interaction with the silver nanoparticles even at low concentrations of the glyphosate. Carbon disulfide is preferably used as a solution of carbon disulfide in dimethyl sulfoxide (DMSO) with a concentration of 4% CS 2 (v / v).

[0012] Silver nanoparticles are used in the form of AgH colloid which is a silver colloid prepared by chemical reduction of the AgNO 3 using NH 2 OH.HCl as a reducing agent; therefore, these are silver plasmonic nanoparticles dispersed in an aqueous environment. The average size of these nanoparticles ranges from 20 to100 nm, the nanoparticles preferably have an average size of 40 nm, and the concentration range of the used silver nanoparticles is about 2x10 11< nanoparticles per 1 ml of water. Besides the amplification of the Raman signal, the silver nanoparticles also act as a catalyst for the formation of the final product in an acidic environment.

[0013] The term alkaline environment for carrying out the chemical reaction of an aqueous solution of glyphosate with carbon disulfide refers to an environment with a pH in the range from 10 to 12, using an aqueous solution of a strong base, such as sodium hydroxide (NaOH) or potassium hydroxide (KOH).

[0014] The term acidic environment in which the adsorption and interaction of carbon disulfide-modified glyphosate with the silver nanoparticles takes place refers to an environment with a pH in the range from 2,5 to 4, wherein an aqueous solution of a strong inorganic acid, such as nitric acid (HNO 3 ), is used.

[0015] The term final product refers to the (5-oxo-(2-thioxothiazolidin-3-yl)methyl)phosphonic acid which can exist in both, oxo- and enol-forms. It has a specific PERS spectrum and corresponds to glyphosate present in the analyzed sample.

[0016] PERS detection of the final product can be carried by a Raman spectrometer known from the state of the art, whereby the corresponding spectrum is recorded for the sample in suspension. The employed radiation is selected from the infrared or visible region of the electromagnetic spectrum, i.e. radiation with a wavelength from 500 nm to 800 nm, monochromatic excitation sources with a longer wavelength are preferably used, such as solid-state diode lasers with the excitation wavelength of 785 nm.

[0017] In addition to the recording of the Raman spectrum of the final product in suspension, it is also possible - before the analysis of the final product in the spectrometer - to drop only microliter volumes (e.g. 1 µl) of the final product formed in the suspension in the presence of silver nanoparticles onto the surface of the nano-optical chip, which was developed by the applicant and is the subject of the international application no. PCT / IB2018 / 060065, published under the International Publication Number WO2019 / 116320A1. This chip consists of a silicon substrate with a deposited layer of gold plasmonic nanoparticles. By dropping the suspension containing both the final product and plasmonic silver nanoparticles onto the surface of the chip a second layer of the nanoparticles is formed which beneficially results in an additional amplification of the PERS signal due to the higher probability of the formation and existence of hot-spots which are responsible for this amplification. The benefit of the use of silicon nano-optical chip, in addition to being portable, is its stability, universality and additional amplification of the measurable signal. Together with the spectrometric equipment, calibration curves and a recorded reference spectrum (developed and supplied by the applicant of this application under the tradenames Ramascope system and Pickmol software, respectively), it also enables an accurate assessment of glyphosate concentrations in the analyzed sample.

[0018] It will be clear to those skilled in the art that the term sample in an aqueous environment refers to an aqueous solution; nevertheless, it can also be any solid or gaseous sample that can be transferred into a solution (e.g. can be dissolved) and therefore, it was not primarily a sample in an aqueous environment.

[0019] For the correct course of the individual reaction processes, it is important to keep the specified pH ranges, while the temperature and pressure are normal - ambient temperature and atmospheric pressure. The ambient temperature refers to a temperature about 20 °C; however, the corresponding method works reliably in the temperature range from 0 °C (the freezing point of water) to 60 °C, as glyphosate and its various forms demonstrate stability only up to a temperature of 60 °C.

[0020] The above-mentioned method can be summarized in the following steps: a) mixing an aqueous solution of a sample containing glyphosate with carbon disulfide in an alkaline environment to form a carbon disulfide-modified glyphosate; b) transferring the carbon disulfide-modified glyphosate obtained in step a) into the AgH colloid in an acidic environment giving rise to a final product, (5-oxo-(2-thioxothiazolidin-3-yl)methyl)phosphonic acid; c) recording of the PERS spectrum of the final product in suspension using a Raman spectrometer and determination of the presence or evaluation of the concentration range of the glyphosate in the sample, or alternatively d) steps a) and b) are carried out and the microliter volumes of the final product from the step b) (5-oxo-(2-thioxothiazolidin-3-yl)methyl)phosphonic acid present in the suspension together with the silver nanoparticles are dropped onto a chip consisting of a silicon substrate with a deposited layer of gold plasmonic nanoparticles, whereas the silver nanoparticles with the final product form a second layer on the top of the layer of gold nanoparticles; and e) recording the PERS spectrum of the final product from the surface of a gold nano-optical chip using a Raman spectrometer and determining the presence or the quantity of glyphosate in the sample.

[0021] A kit for carrying out the method of detection of glyphosate in samples in an aqueous environment as described above is also described, and the kit comprises: a vial for a carbon disulfide solution in an alkaline environment with a pH in the range of 10 to 12 and the corresponding reagents for its preparation; a vial for a solution of AgH colloid in an acidic environment with a pH in the range of 2,5 to 4 and the corresponding reagents for its preparation; Raman spectrometer; optionally a silicon chip with a deposited layer of gold plasmonic nanoparticles; and manual.

[0022] Due to the fact that the acidic AgH colloid does not remain stable for a long time, it is necessary to provide the appropriate reagents for its preparation in the kit and thus, to prepare the colloid with the required pH only shortly before the glyphosate detection itself. At the same time, there is also a vial in the kit provided, optionally a cuvette, for recording the PERS spectrum of the final product in a suspension, whereas it also serves for the preparation of the colloid with the required characteristics at an appropriate moment.

[0023] Similarly, a solution of carbon disulfide in an alkaline environment with the required pH is also prepared shortly before the glyphosate detection itself. Therefore, the kit also provides the corresponding reagents for its preparation as well as an appropriate vial for these purposes.

[0024] Even though the present method is not a direct glyphosate detection method and thus a modification of the sample is needed, this sample modification is simple, time- and cost-effective compared to sample (pretreatment required in the state-of-the-art glyphosate detection methods.Brief description of drawings

[0025] Figure 1Schematic representation of the method for the detection of glyphosate in samples in an aqueous environment according to the present invention; Figure 2Presumed simplified mechanism of formation of the final product 5, or 6; Figure 3 A Raman spectrum of glyphosate in a solid state, in aqueous solution and in a suspension of silver plasmonic nanoparticles, and PERS spectrum characteristic for the final product. B An example of the corresponding calibration curve demonstrating the dependence of the signal intensity on the increasing concentration of glyphosate in the concentration range 0 - 6,0x10 -6< M, detection limit (LOD) is about 1 ppb.

[0026] The following examples serve to illustrate the present invention; however, the invention should not be limited to them. Those skilled in the art will be able to perform various modifications, variations and changes without departing from the scope of the present application (as defined by the claims). The interpretation of the scope of the claims should be interpreted as a whole and in broadest scope consistent with the specification which is not limited to the specific examples provided.ExamplesExample 1Detection of glyphosate in a suspension

[0027] In accordance with Figure 1, 1 ml of an aqueous solution of the sample suspected to contain glyphosate 1 was added to a glass tube containing 50 µl of a carbon disulfide solution in DMSO with a concentration of 4% CS 2 (v / v) in an alkaline environment consisting of 1 M aqueous solution of NaOH (5 µl), with a pH of the reaction mixture about 11, whereas mixing these solutions resulted in the formation of carbon disulfide-modified glyphosate 2. The above-mentioned corresponds to step a) in Figure 1. In this step, the amino group of glyphosate 1 is protected by the reaction of the molecule with carbon disulfide in an alkaline environment. The resulting product, referred to in the previous text as carbon disulfide-modified glyphosate 2, is N-dithiocarboxy-N-(phosphonomethyl)glycine 2, which contains -SH (thiol) and C=S (thio-) functional groups in its structure, which play a major role in a subsequent step b) according to Figure 1. In this step, 10 µl of carbon disulfide-modified glyphosate 2 obtained in step a) was transferred to a glass cuvette containing 1 ml of AgH colloid (prepared by chemical reduction of AgNO 3 using NH 2 OH.HCl as a reducing agent) containing silver nanoparticles with an average size of 40 nm dispersed in aqueous solution, at a concentration of about 2x10 11< nanoparticles per 1 ml of water, with a pH about 2,5 formed by the addition of 20 µl of a 0,1 M aqueous solution of nitric acid. The incorporated -SH and C=S groups of carbon disulfide-modified glyphosate 2 demonstrate a high affinity towards the silver nanoparticle surface, which enables adsorption of carbon disulfide-modified glyphosate molecule onto the surface of plasmonic silver nanoparticles - such a molecule 3 adsorbed onto the surface of nanoparticles is represented by the reference number 3 in Figure 1. Silver nanoparticles of the AgH colloid also act as a catalyst for the subsequent cyclization of molecule 3 to the final product in an acidic environment, (5-oxo-(2-thioxothiazolidin-3-yl)methyl)phosphonic acid 5, which can exist in oxo- 5 and enol- 6 form. The transition state of molecule 3 to the cyclic form is shown in Figure 1 by the structure marked with reference number 4. Reference numbers 3 and 4 refer to transitional structures (intermediate products) by which it is to be demonstrated the proposed mechanism of formation of the final product 5 or 6. Subsequently, the presence of glyphosate is detected by recording the PERS spectrum of the final product in suspension using a Raman spectrometer - thereby one determines its presence or absence in the examined sample. Based on the evaluation of the intensity of the recorded spectrum, it is possible to perform an initial assessment of the concentration range of the herbicide glyphosate present in the sample.

[0028] The given assumed simplified mechanism for the formation of final products 5 or 6, as described above is depicted in Figure 2, whereas used reference numbers 1-6 correspond to the reference numbers in Figure 1.Example 2Detection of glyphosate after its application onto the surface of a gold nano-optical chip

[0029] Aqueous solution of the sample is processed in the same way as in Example 1, with a difference that the PERS spectrum of the final product is not recorded in suspension, but microliter volumes (1 µl) of the suspension is dropped onto a chip consisting of a silicon substrate with a deposited layer of gold plasmonic nanoparticles, whereas the silver nanoparticles with the final product form a second layer on the top of the layer of gold nanoparticles. The above-mentioned corresponds to step d) in Figure 1. The silicon substrate is a silicon wafer with dimensions of 5x5x0,5 mm. A layer of gold nanoparticles is deposited on this silicon wafer by sputtering process with preferred thickness of 50 nm. Transferring the suspension of the final product to the chip can be done by dropping a certain amount of the suspension (e.g. 1µl) onto the chip. Subsequently, the presence of glyphosate is detected by recording the PERS spectrum of the final product from the surface of the chip using a Raman spectrometer - thereby one determines its presence or absence in the examined sample. Based on the evaluation of the intensity of the recorded spectrum, it is possible to determine quantity of the glyphosate in the sample.List of reference numbers

[0030] 1 glyphosate 2 carbon disulfide-modified glyphosate (N-dithiocarboxy-N-(phosphonomethyl)glycine) 3 molecule of the carbon disulfide-modified glyphosate adsorbed on the surface of plasmonic silver nanoparticles 4 molecule of the carbon disulfide-modified glyphosate in the process of cyclization catalyzed by silver nanoparticles 5 final product ((5-oxo-(2-thioxothiazolidin-3-yl)methyl)phosphonic acid) in oxo- form 6 final product ((5-oxo-(2-thioxothiazolidin-3-yl)methyl)phosphonic acid) in enol- form Industrial applicability

[0031] The present invention, which relates to a method for detection of the herbicide glyphosate in water using PERS spectroscopy and chemical modification of the molecule catalyzed by silver plasmonic nanoparticles since direct glyphosate detection using silver nanoparticles is significantly limited allows detecting glyphosate at low concentrations, whereas identification of the glyphosate is carried out on the basis of the specific PERS spectrum, i.e. it is a highly sensitive and selective detection method. In addition, the relative intensity of the recorded Raman (PERS) signal allows the use of this fast and soft screening analysis for an initial assessment of the concentration range of the herbicide present, or the determination of its quantity.

Claims

1. Method for selective and sensitive detection of glyphosate using plasmon-enhanced Raman spectroscopy, characterized in that it comprises the following steps: a) mixing an aqueous solution of a sample containing glyphosate with carbon disulfide in an alkaline environment to form a carbon disulfide-modified glyphosate; b) transferring the carbon disulfide-modified glyphosate obtained in step a) into the AgH colloid in an acidic environment giving rise to a final product, (5-oxo-(2-thioxothiazolidin-3-yl)methyl)phosphonic acid; c) detection of glyphosate and recording of the PERS spectrum of the final product in suspension using a Raman spectrometer and determination of the presence and / or evaluation of the concentration range of the glyphosate in the sample, or alternatively d) steps a) and b) are carried out and the microliter volumes of the final product from the step b) (5-oxo-(2-thioxothiazolidin-3-yl)methyl)phosphonic acid are dropped onto a chip consisting of a silicon substrate with a deposited layer of gold plasmonic nanoparticles, whereas the silver nanoparticles with the final product form a second layer on the top of the layer of gold nanoparticles; and e) detection of glyphosate by recording the PERS spectrum of the final product from the surface of a gold nano-optical chip using a Raman spectrometer and determining the presence and / or the quantity of glyphosate in the sample.

2. Method according to claim 1, characterized in that the alkaline environment in step a) has a pH in the range from 10 to 12.

3. Method according to any of the preceding claims, characterized in that the alkaline environment in step a) refers to an aqueous solution of a strong base, preferably a 1M aqueous solution of sodium hydroxide.

4. Method according to any of the preceding claims, characterized in that carbon disulfide is used as a solution of carbon disulfide in dimethyl sulfoxide (DMSO) with a concentration of 4% CS2 (v / v).

5. Method according to any of the preceding claims, characterized in that pH of the acidic environment in step b) is in the range from 2,5 to 4,0.

6. Method according to any of the preceding claims, characterized in that AgH colloid consists of Ag nanoparticles dispersed in aqueous environment and having average nanoparticle size in the range from 20 to 100 nm, preferably 40 nm.

7. Method according to claim 6, characterized in that the concentration of the Ag nanoparticles in AgH colloid is about 2x1011 nanoparticles per 1 ml of water.

8. Method according to any of the preceding claims, characterized in that the acidic environment in the step b) refers to an aqueous solution of a strong inorganic acid, preferably a 0,1M aqueous solution of nitric acid.

9. Method according to any of the preceding claims, characterized in that detection of the final product, (5-oxo-(2-thioxothiazolidin-3-yl)methyl)phosphonic acid, using a Raman spectrometer is carried out at the wavelength of the radiation selected from the infrared or visible region of the electromagnetic spectrum.

10. Use of the method according to any of the preceding claims for determining the presence and / or quantity, or evaluating the concentration range of the glyphosate in the analysed sample.