New Spectrophotometric and Electrochemical Enzyme Biosystem Based on Laccase and Ionic Liquid for the Detection of Glyphosate in Biological Samples

Galai S*, Omar S, HajKacem S, Aladnani A, Rosatella A, Antonia PLR, Fernandez FJH, Afonso C5, Diouani MF

**Galai S*, Omar S, HajKacem S, Aladnani A, Rosatella A, Antonia PLR, Fernandez FJH, Afonso C5, Diouani MF**

Volume6-Issue10
Dates: Received: 2025-10-04 | Accepted: 2025-10-29 | Published: 2025-10-30
Pages: 1614-1625

Abstract

Glyphosate (N- (Phosphonomethyl) glycine), one of the most widely used pesticides in the world. The detection methods are difficult to implement, time consuming and expensive due to its chemical properties and its low prevalence; there is a strong need to develop quick-sensitive analytical methods for Glyphosate (GLP) assay and monitoring. For this purpose, a new biosystem based on enzyme reaction was implemented by laccase, redox mediator (Acetosyringone: ASGN), and ionic liquid (IL, as conservator and activator) to catalyse GLP. The laccase-catalytic system has been investigated by two analytical methods: spectrophotometric and electrochemical one. In terms of efficiency, the detection limit for spectrophotometric method was 25 μM GLP, while electrochemical method was even lowest around 5 μM GLP. The developing biosensor based on this enzymatic system has been carried out using gold-plated screen-printed electrode and Nafion polymer for laccase, redox mediator and ionic liquid complex immobilization. GLP samples were successfully analyzed using Cyclic Voltammetry (CV) measurement at scan rate of 100 mV/s. The concentration of GLP was accurately determined in the range of 5 μM to 15 μM GLP, and high correlation rate (98%) between current density and GLP concentration was determined using the laccase-based-biosensor, which shown good reproducibility and repeatability, high selectivity and therefore it has been used for GLP assays in biological samples (Cell lysate and culture medium).

FullText HTML FullText PDF DOI: 10.37871/jbres2215

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How to cite this article

Galai S, Omar S, HajKacem S, Aladnani A, Rosatella A, Antonia PLR, Fernandez FJH, Afonso C, Diouani MF. New Spectrophotometric and Electrochemical Enzyme Biosystem Based on Laccase and Ionic Liquid for the Detection of Glyphosate in Biological Samples. J Biomed Res Environ Sci. 2025 Oct 30; 6(10): 1614-1625. doi: 10.37871/jbres2215, Article ID: JBRES2215, Available at: https://www.jelsciences.com/articles/jbres2215.pdf

Subject area(s)

Biochemistry

Bioengineering

Toxicology

References

Roberts CW, Roberts F, Lyons RE, Kirisits MJ, Mui EJ, Finnerty J, Johnson JJ, Ferguson DJ, Coggins JR, Krell T, Coombs GH, Milhous WK, Kyle DE, Tzipori S, Barnwell J, Dame JB, Carlton J, McLeod R. The shikimate pathway and its branches in apicomplexan parasites. J Infect Dis. 2002 Feb 15;185 Suppl 1:S25-36. doi: 10.1086/338004. PMID: 11865437.
Abraham W. Glyphosate formulations and their use for the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase. US Patent. 2010:7771736.
Larsen K, Najle R, Lifschitz A, Maté ML, Lanusse C, Virkel GL. Effects of Sublethal Exposure to a Glyphosate-Based Herbicide Formulation on Metabolic Activities of Different Xenobiotic-Metabolizing Enzymes in Rats. Int J Toxicol. 2014 Jul;33(4):307-318. doi: 10.1177/1091581814540481. Epub 2014 Jul 1. PMID: 24985121.
Ford B, Bateman LA, Gutierrez-Palominos L, Park R, Nomura DK. Mapping Proteome-wide Targets of Glyphosate in Mice. Cell Chem Biol. 2017 Feb 16;24(2):133-140. doi: 10.1016/j.chembiol.2016.12.013. Epub 2017 Jan 26. PMID: 28132892.
Reddy KN, Rimando AM, Duke SO. Aminomethylphosphonic acid, a metabolite of glyphosate, causes injury in glyphosate-treated, glyphosate-resistant soybean. J Agric Food Chem. 2004 Aug 11;52(16):5139-43. doi: 10.1021/jf049605v. PMID: 15291487.
Castellani R, Smith MA, Richey PL, Perry G. Glycoxidation and oxidative stress in Parkinson disease and diffuse Lewy body disease. Brain Res. 1996 Oct 21;737(1-2):195-200. doi: 10.1016/0006-8993(96)00729-9. PMID: 8930366.
Hawkins M. Updated review of glyphosate (103601). Incident reports. memorandum, EPA Toxicology and Epidemiology Branch. 2009.
Çetin E, Şahan S, Ülgen A, Şahin U. DLLME-spectrophotometric determination of glyphosate residue in legumes. Food Chem. 2017 Sep 1;230:567-571. doi: 10.1016/j.foodchem.2017.03.063. Epub 2017 Mar 14. PMID: 28407950.
Kataoka H, Ryu S, Sakiyama N, Makita M. Simple and rapid determination of the herbicides glyphosate and glufosinate in river water, soil and carrot samples by gas chromatography with flame photometric detection. J Chromatogr A. 1996;726(1-2):253-8. doi: 10.1016/0021-9673(95)01071-8.
Aguirre M del C, Urreta SE, Gomez CG. A Cu2+-Cu/glassy carbon system for glyphosate determination. Sens Actuators B-Chem. 2019;284:675-83. doi: 10.1016/j.snb.2018.12.124.
Demonte LD, Michlig N, Gaggiotti M, Adam CG, Beldoménico HR, Repetti MR. Determination of glyphosate, AMPA and glufosinate in dairy farm water from Argentina using a simplified UHPLC-MS/MS method. Sci Total Environ. 2018 Dec 15;645:34-43. doi: 10.1016/j.scitotenv.2018.06.340. Epub 2018 Jul 14. PMID: 30015116.
Fabbrini M, Galli C, Gentili P. Radical or electron-transfer mechanism of oxidation with some laccase/mediator systems. J Mol Catal B Enzym. 2002 : doi:10.1016/S1381-1177(02)00082-6.
Zaccaria M, Dawson W, Russel Kish D, Reverberi M, Bonaccorsi di Patti MC, Domin M, Cristiglio V, Chan B, Dellafiora L, Gabel F, Nakajima T, Genovese L, Momeni B. Experimental-theoretical study of laccase as a detoxifier of aflatoxins. Sci Rep. 2023 Jan 17;13(1):860. doi: 10.1038/s41598-023-27519-1. PMID: 36650163; PMCID: PMC9845376.
Welton T. Room-Temperature Ionic Liquids. Solvents for Synthesis and Catalysis. Chem Rev. 1999 Aug 11;99(8):2071-2084. doi: 10.1021/cr980032t. PMID: 11849019.
Galai S, de los Ríos AP, Hernández-Fernández FJ, Kacem SH, Tomas-Alonso F. Over-activity and stability of laccase using ionic liquids: screening and application in dye decolorization. RSC Adv. 2015;5(21):16173-89. doi: 10.1039/C4RA07351G.
Freemantle M. An introduction to ionic liquids. Cambridge, England: Royal Society of Chemistry; 2010.
Pizzul L, Castillo Mdel P, Stenström J. Degradation of glyphosate and other pesticides by ligninolytic enzymes. Biodegradation. 2009 Nov;20(6):751-9. doi: 10.1007/s10532-009-9263-1. Epub 2009 Apr 25. PMID: 19396551.
Valle AL, Mello FCC, Alves-Balvedi RP, Rodrigues LP, Goulart LR. Glyphosate detection: methods, needs and challenges. Environ Chem Lett. 2019;17(1):291-317. doi: 10.1007/s10311-018-0789-5.
Pollegioni L, Schonbrunn E, Siehl D. Molecular basis of glyphosate resistance-different approaches through protein engineering. FEBS J. 2011 Aug;278(16):2753-66. doi: 10.1111/j.1742-4658.2011.08214.x. Epub 2011 Jun 28. PMID: 21668647; PMCID: PMC3145815.
Martin E, Dubessay P, Record E, Audonnet F, Michaud P. Recent advances in laccase activity assays: A crucial challenge for applications on complex substrates. Enzyme Microb Technol. 2024 Feb;173:110373. doi: 10.1016/j.enzmictec.2023.110373. Epub 2023 Dec 10. PMID: 38091836.
Cohen R, Persky L, Hadar Y. Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus. Appl Microbiol Biotechnol. 2002 Apr;58(5):582-94. doi: 10.1007/s00253-002-0930-y. Epub 2002 Feb 15. PMID: 11956739.
Tiwari A, Chen CW, Haldar D, Patel AK, Dong CD, Singhania RR. Laccase in biorefinery of lignocellulosic biomass. Appl Sci. 2023;13:4673. doi: 10.3390/app13084673.
Said G, Hafsa KY, Nejib MM. Characterization of yellow bacterial laccase SmLac / role of redox mediators in azo dye decolorization. Journal of Chemical Technology & Biotechnology. 2013. doi: 10.1002/jctb.4254.
Mendoza L, Jonstrup M, Hatti-Kaul R, Mattiasson B. Azo dye decolorization by a laccase/mediator system in a membrane reactor: enzyme and mediator reusability. Enzyme Microb Technol. 2011 Oct 10;49(5):478-84. doi: 10.1016/j.enzmictec.2011.08.006. Epub 2011 Aug 27. PMID: 22112621.
Sihem HK, Said G, de los Ríos AP, José FFH, Issam S. New efficient laccase immobilization strategy using ionic liquids for biocatalysis and microbial fuel cells applications: New polymeric ionic liquid laccase membrane. J Chem Technol Biotechnol. 2018;93(1):174-83.
Button DK, Robertson B, Gustafson E, Zhao X. Experimental and theoretical bases of specific affinity, a cytoarchitecture-based formulation of nutrient collection proposed to supercede the Michaelis-Menten paradigm of microbial kinetics. Appl Environ Microbiol. 2004 Sep;70(9):5511-21. doi: 10.1128/AEM.70.9.5511-5521.2004. PMID: 15345439; PMCID: PMC520905.
Ehrl BN, Mogusu EO, Kim K, Hofstetter H, Pedersen JA, Elsner M. High Permeation Rates in Liposome Systems Explain Rapid Glyphosate Biodegradation Associated with Strong Isotope Fractionation. Environ Sci Technol. 2018 Jul 3;52(13):7259-7268. doi: 10.1021/acs.est.8b01004. Epub 2018 Jun 19. PMID: 29790342; PMCID: PMC7193547.
Songa EA, Waryo T, Jahed N, Baker PGL, Kgarebe BV, Iwuoha EI. Electrochemical nanobiosensor for glyphosate herbicide and its metabolite. Electroanalysis. 2009;21(3-5):671-4.
Oliveira GC, Moccelini SK, Castilho M, Terezo AJ, Possavatz J, Magalhães MR, Dores EF. Biosensor based on atemoya peroxidase immobilised on modified nanoclay for glyphosate biomonitoring. Talanta. 2012 Aug 30;98:130-6. doi: 10.1016/j.talanta.2012.06.059. Epub 2012 Jul 1. PMID: 22939138.
Nguyen HH, Lee SH, Lee UJ, Fermin CD, Kim M. Immobilized Enzymes in Biosensor Applications. Materials (Basel). 2019 Jan 2;12(1):121. doi: 10.3390/ma12010121. PMID: 30609693; PMCID: PMC6337536.