Glyphosate is the most widespread herbicide and its global use is steadily increasing. Although glyphosate is considered to have low toxicity, its wide application has raised concerns about its effects on human health. The extensive use of glyphosate has risen a need of its continuous monitoring in drinking and surface waters to assure in accordance with the set standards. Within the present study, we have developed a novel assay for the on-site detection of glyphosate by combining flow-through technology with the high specificity of immunorecognition. The proposed biosensing system was based on the detection of fluorescence signal generated by the quantitative replacement of glyphosate in antigen-antibody complex with IgY-type anti-glyphosate antibodies on microbeads by synthetic 5-carboxytetramethylrhodamine (5-TAMRA) conjugated glyphosate. The working range of this assay was in low millimolar range and the time required for glyphosate detection around 0.5 h. The applicability of the immunoassay for glyphosate detection in surface water was tested and the biosensor results were validated with high-performance liquid chromatography.
This is a preview of subscription content,to check access.
Access this article
Similar content being viewed by others
Aneja, A., Mathur, N., Bhatnagar, P. K., & Mathur, P. C. (2008). Triple-FRET technique for energy transfer between conjugated polymer and TAMRA dye with possible applications in medical diagnostics. Journal of Biological Physics, 34(5), 487–493.
Bhaskara, B., & Nagaraja, P. (2006). Direct sensitive spectrophotometric determination of glyphosate by using ninhydrin as a chromogenic reagent in formulations and environmental water samples. HCA, 89(11), 2686–2693.
Bhullar, B. S. (1998). Linker-assisted immunoassay for glyphosate. Patent WO2000014538A1.
Clegg, B. S., Stephenson, G. R., & Hall, J. C. (1999). Development of an enzyme-linked immunosorbent assay for the detection of glyphosate. Journal of Agricultural and Food Chemistry, 47(12), 5031–5037.
Cooper, M., Ebner, A., Briggs, M., Burrows, M., Gardner, N., Richardson, R., & West, R. (2004). Cy3BTM: Improving the performance of cyanine dyes. Journal of Fluorescence, 14(2), 145–150.
Coupe, R. H., Kalkhoff, S. J., & Capel, P. D. (2011). Fate and transport of glyphosate and aminomethylphoshonic acid in surface waters of agricultural basin. Pest Management Science, 68(1), 16–30.
Ding, X., & Yang, K. L. (2013). Development of an oligopeptide functionalized surface plasmon resonance biosensor for online detection of glyphosate. Analytical Chemistry, 85(12), 5727–5733.
Duke, S. O., & Powles, S. B. (2008). Glyphosate: a once in a century herbicide. Pest Management Science, 64, 319–325.
EC. European Commission. (1998). Council Directive 98/83/EC on the quality of water intended for human consumption. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:1998:330:0032:0054:EN:PDF. Accessed 16 Jan 2019.
EC. European Commission. (2018). Pesticides Database. http://ec.europa.eu/sanco_pesticides/public/index.cfm. Accessed 16 Jan 2019.
EFSA. European Food Safety Authority. (2015). Glyphosate: EFSA updates toxicological profile. http://www.efsa.europa.eu/en/press/news/151112. Accessed 16 Jan 2019.
Ehlers, J. E., Rondan, N. G., Huynh, L. K., Pham, H., Marks, M., & Truong, T. N. (2007). Theoretical study on mechanisms of the epoxy-amine curing reaction. Macromolecules, 40(12), 4370–4377.
FAO/WHO. Food and Agriculture Organization of the United Nations/World Health Organization. (2019). CODEX Alimentarius International Food Standards. http://www.fao.org/fao-who-codexalimentarius/codex-texts/dbs/pestres/pesticide-detail/en/?p_id=158. Accessed 16 Jan 2019.
FAO/WHO Meeting on Pesticide Residues. (1997). Pesticide residues in food 1997. http://www.inchem.org/documents/jmpr/jmpmono/v097pr04.htm. Accessed 16 Jan 2019.
FAO/WHO Meeting on Pesticide Residues. (2016). Pesticide residues in food 2016. http://www.fao.org/3/a-i5693e.pdf. Accessed 16 Jan 2019.
Giesy, J. P., Stuart, D., & Solomon, K. R. (2000). Ecotoxicological risk assessment for Roundup® herbicide. Reviews of Environmental Contamination and Toxicology, 167, 35–120.
Glass, R. L. (1981). Colorimetric determination of glyphosate in water after oxidation to orthophosphate. Analytical Chemistry, 53(6), 921–923.
González-Martínez, M. Á., Brun, E. M., Puchades, R., Maquieira, Á., Ramsey, K., & Rubio, F. (2005). Glyphosate immunosensor. Application for water and soil analysis. Analytical Chemistry, 77(13), 4219–4227.
Government of Canada. (2017). Canadian Drinking Water Guidelines. https://www.canada.ca/en/health-canada/services/environmental-workplace-health/reports-publications/water-quality/guidelines-canadian-drinking-water-quality-summary-table.html. Accessed 16 Jan 2019.
Guyton, K. Z., Loomis, D., Grosse, Y., El Ghissassi, F., Benbrahim-Tallaa, L., Guha, N., Scoccianti, C., Mattock, H., & Straif, K. (2015). Carcinogenicity of tetrachlorvinphos, parathion, malathion, diazinon, and glyphosate. The Lancet Oncology, 16(5), 490–491.
Hamilton, D. J., Ambrus, A., Dieterle, R. M., Felsot, A. S., Harris, C. A., Holland, P. T., Katayama, A., Kurihara, N., Linders, J., Unsworth, J., & Wong, S.-S. (2003). Regulatory limits for pesticide residues in water (IUPAC Technical Report). Pure and Applied Chemistry, 75(8), 1123–1155.
Henderson, A. M., Gervais, J. A., Luukinen, B., Buhl, K., & Stone, D. (2015). Glyphosate technical fact sheet. Corvallis: National Pesticide Information Center, Oregon State University Extension http://npic.orst.edu/factsheets/archive/glyphotech.html. Accessed 16 Jan 2019.
Hensle, E. M., Esfandiari, N. M., Lim, S., & Blum, S. A. (2014). BODIPY fluorophore toolkit for probing chemical reactivity and for tagging reactive functional groups. European Journal of Organic Chemistry, 2014(16), 3347–3354.
Juronen, D., Kuusk, A., Kivirand, K., Rinken, A., & Rinken, T. (2018). Immunosensing system for rapid multiplex detection of mastitis-causing pathogens in milk. Talanta, 178, 949–954.
Lee, E. A., Zimmerman, L. R., Bhullar, B. S., & Thurman, E. M. (2002). Linker-assisted immunoassay and liquid chromatography/mass spectrometry for the analysis of glyphosate. Analytical Chemistry, 74(19), 4937–4943.
Lee, H. U., Shin, H. Y., Lee, J. Y., Song, Y. S., Park, C., & Kim, S. W. (2010). Quantitative detection of glyphosate by simultaneous analysis of UV spectroscopy and fluorescence using DNA-labeled gold nanoparticles. Journal of Agricultural and Food Chemistry, 58(23), 12096–12100.
Lee, H. U., Jung, D. U., Lee, J. H., Song, Y. S., Park, C., & Kim, S. W. (2013). Detection of glyphosate by quantitative analysis of fluorescence and single DNA using DNA-labeled fluorescent magnetic core-shell nanoparticles. Sensors and Actuators B, 177, 879–886.
Luijendijk, C. D., Beltman, W. H. J., & Wolters, M. F. (2003). Measures to reduce glyphosate runoff from hard surfaces. Wageningen: Plant Research International B.V.
Martínez Gil, P., Laguarda-Miro, N., Camino, J. S., & Peris, R. M. (2013). Glyphosate detection with ammonium nitrate and humic acids as potential interfering substances by pulsed voltammetry technique. Talanta, 115, 702–705.
Mörtl, M., Németh, G. Č., Juracsek, J., Darvas, B., Kamp, L., Rubio, F., & Sézkács, A. (2013). Determination of glyphosate residues in Hungarian water samples by immunoassay. Microchemical Journal, 107, 143–151.
Myers, J. P., Antoniou, M. N., et al. (2018). Concerns over use of glyphosate-based herbicides and risks associated with exposures: a consensus statement. Environmental Health, 15(19), 1–13.
Oliveira, G. C., Moccelini, S. K., Castilho, M., Terezo, A. J., Possavatz, J., Magalhães, M. R. L., & Dores, E. F. G. C. (2012). Biosensor based on atemoya peroxidase immobilised on modified nanoclay for glyphosate biomonitoring. Talanta, 98, 130–136.
Peedel, D., & Rinken, T. (2014). Rapid biosensing of Staphylococcus aureus bacteria in milk. Analytical Methods, 6(8), 2642–2647.
Rinken, A., Lavogina, D., & Kopanchuk, S. (2018). Assays with detection of fluorescence anisotropy: challenges and possibilities for characterizing ligand binding to GPCRs. Trends in Pharmacological Sciences, 39(2), 187–199.
Rojano-Delgado, A. M., Ruiz-Jiménez, J., de Castro, M. D., & De Prado, R. (2010). Determination of glyphosate and its metabolites in plant material by reversed-polarity CE with indirect absorptiometric detection. Electrophoresis, 31(8), 1423–1430.
Romieu, A., Massif, C., Rihn, S., Ulrich, G., Ziessel, R., & Renard, P. Y. (2013). The first comparative study of the ability of different hydrophilic groups to water-solubilise fluorescent BODIPY dyes. New Journal of Chemistry, 37(4), 1016–1027.
Royer, A., Beguin, S., Tabet, J. C., Hulot, S., Reding, M. A., & Communal, P. Y. (2000). Determination of glyphosate and aminomethylphosphonic acid residues in water by gas chromatography with tandem mass spectrometry after exchange ion resin purification and derivatization. Application on vegetable matrixes. Analytical Chemistry, 72(16), 3826–3832.
Rubio, F., Veldhuis, L. J., Clegg, B. S., Fleeker, J. R., & Hall, J. C. (2003). Comparison of a direct ELISA and an HPLC method for glyphosate determinations in water. Journal of Agricultural and Food Chemistry, 51(3), 691–696.
Silva, E. R., Segato, T. P., Coltro Wendell, K. T., Lima, R. S., Carrilho, E., & Mazo, L. H. (2013). Determination of glyphosate and AMPA on polyester-toner electrophoresis microchip with contactless conductivity detection. Electrophoresis, 34, 2107–2111.
Songa, E. A., Arotiba, O. A., Owino, J. H. O., Jahed, N., Baker, P. G. L., & Iwuoha, E. I. (2009a). Electrochemical detection of glyphosate herbicide using horseradish peroxidase immobilized on sulfonated polymer matrix. Bioelectrochemistry, 75(2), 117–123.
Songa, E., Waryo, T., Jahed, N., Baker, P., Kgarebe, B., & Iwuoha, E. (2009b). Electrochemical nanobiosensor for glyphosate herbicide and its metabolite. Electroanalysis, 21(3-5), 671–674.
Stalikas, C. D., & Konidari, C. N. (2001). Analytical methods to determine phosphonic and amino acid group-containing pesticides. Journal of Chromatography. A, 907(1-2), 1–19.
Todorovic, G. R., Rampazzo, N., Mentler, A., Blum, W. E. H., Eder, A., & Strauss, P. (2014). Influence of soil tillage and erosion on the dispersion of glyphosate and aminomethylphosphonic acid in agricultural soils. International Agrophysics, 28, 93–100.
Torstensson, L., Börjesson, E., & Stenström, J. (2005). Efficacy and fate of glyphosate on Swedish railway embankments. Pest Management Science, 61(9), 881–886.
Tu, M., Hurd, C. & Randall, J.M. (2001). Weed Control Methods Handbook: Tools and Techniques for Use in Natural Areas, http://tncweeds.ucdavis.edu. Accessed 16 Jan 2019.
USDA. United States Department of Agriculture. (2018). International Maximum Residue Level Database. www.mrldatabase.com/. Accessed 16 Jan 2019.
USGS. U.S. Geological Survey. (2015). Estimated Annual Agricultural Pesticide Use. https://water.usgs.gov/nawqa/pnsp/usage/maps/show_map.php?year=2015&map=GLYPHOSATE&hilo=L&disp=Glyphosate. Accessed 16 Jan 2019.
Van Bruggen, A. H. C., He, M. M., Shin, K., Mai, V., Jeong, K. C., Finckh, M. R., & Morris, J. G. (2018). Environmental and health effects of the herbicide glyphosate. Science of the Total Environment, 616-617, 255–268.
Veiksina, S., Kopanchuk, S., & Rinken, A. (2010). Fluorescence anisotropy assay for pharmacological characterization of ligand binding dynamics to melanocortin 4 receptors. Analytical Biochemistry, 402(1), 32–39.
Veiksina, S., Kopanchuk, S., & Rinken, A. (2014). Budded baculoviruses as a tool for a homogeneous fluorescence anisotropy-based assay of ligand binding to G protein-coupled receptors: the case of melanocortin 4 receptors. Biochimica et Biophysica Acta, 1838(1 Pt B), 372–381.
Wang, D., Lin, B., Cao, Y., Guo, M., & Yu, Y. (2016). A highly selective and sensitive fluorescence detection method of glyphosate based on an immune reaction strategy of carbon dot labeled antibody and antigen magnetic beads. Journal of Agricultural and Food Chemistry, 64(30), 6042–6050.
Zheng, J., Zhang, H., Qu, J., Zhu, Q., & Chen, X. (2013). Visual detection of glyphosate in environmental water samples using cysteamine-stabilized gold nanoparticles as colorimetric probe. Analytical Methods, 5(4), 917–924.
The authors would like to thank Dr. Riin Rebane from the Estonian Environmental Research Center for her contribution to the validation of biosensor results.
This work was supported by the Estonian Research Council Grant No. IUT 20-17.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
1H NMR spectrum of N-(hept-6-ynoyl)-N-(phosphonomethyl)glycine. (DOCX 163 kb)
13C NMR spectrum of N-(hept-6-ynoyl)-N-(phosphonomethyl)glycine. (DOCX 104 kb)
1H-1H COSY NMR spectrum of N-(hept-6-ynoyl)-N-(phosphonomethyl)glycine. (DOCX 113 kb)
1H-13C HSQC NMR spectrum of N-(hept-6-ynoyl)-N-(phosphonomethyl)glycine. (DOCX 95 kb)
1H-13C HMBC NMR spectrum of N-(hept-6-ynoyl)-N-(phosphonomethyl)glycine. (DOCX 107 kb)
HPLC-MS spectra of 5-TAMRA-glyphosate. From above: a.) Ion chromatogram with TIC, m/z=396 and m/z=788, b.) Extracted mass spectrum from 10,52 min datapoint, c.) Chromatogram at wavelength 280 nm, d.) Chromatogram at wavelength 546 nm. (DOCX 170 kb)
About this article
Cite this article
Viirlaid, E., Ilisson, M., Kopanchuk, S. et al. Immunoassay for rapid on-site detection of glyphosate herbicide. Environ Monit Assess 191, 507 (2019). https://doi.org/10.1007/s10661-019-7657-z