Green Peptide-Assisted Synthesis of Gold Nanoparticles for Electrochemical Biosensing of Carbamate Pesticides

Marcos R. de A. Silva; Barbara B. Gerbelli; Ana Cristina H. Castro-Kochi; Andrea M. Aguilar; Wendel A. Alves

Abstract

Pest-control chemicals are widely used to increase agricultural productivity; however, their extensive application raises concerns regarding food safety, occupational health, and environmental contamination. Therefore, the development of efficient and reliable technologies for pesticide detection, particularly in environmental samples, remains a key research priority. In this study, an electrochemical biosensor based on square-wave voltammetry was developed by immobilizing acetylcholinesterase onto hydrothermally synthesized gold nanoparticles, using the cysteine–diphenylalanine (CFF) peptide as both a reducing and stabilizing agent. The CFF peptide enabled excellent morphological control of the gold nanostructures (hydrodynamic radius, 16 nm) and a uniform size distribution (polydispersity index, PDI = 0.322). Carbamate detection was performed by square-wave voltammetry, yielding a highly sensitive analytical response. The calibration curve exhibited a linear range up to 10^-9 M, with a correlation coefficient (R²) of 0.99 and a detection limit of 0.94 nM. The use of the CFF peptide and its self-assembling properties enabled the fabrication of an efficient and low-cost biosensor for carbamate detection, representing a promising approach for future environmental and agricultural monitoring applications.

References

1.
Xiumei, H.; Vimal, K, B.; Vanloon, W.G.; et al. Degradation of the Pesticide Fenitrothion as Mediated by Cationic Surfactants and α-Nucleophilic Reagents. Langmuir 2006, 22, 9009–9017.
2.
Scognamiglio, V.; Rea, G.; Arduini, F.; et al. Biosensors for Sustainable Food-New Opportunities and Technical Challenges; Elsevier: Amsterdam, The Netherlands, 2016; Volume 74, pp. 3–342.
3.
Gerbelli, B.B.; Sodre, P.T.; Filho, P.L.O.; et al. Enhancing pesticide detection: The role of serine in lipopeptide nanostructures and their self-assembly dynamics. J. Colloid Interface Sci. 2025, 690, 137271.
4.
Gerbelli, B.B.; Filho, P.L.O.; Cortez, B.; et al. Interaction between glyphosate pesticide and amphiphilic peptides for colorimetric analysis. Nanoscale Adv. 2022, 4, 3592–3599.
5.
Tankiewicz, M.; Fenik, J.; Biziuk, M. Determination of organophosphorus and organonitrogen pesticides in water samples. TrAC Trends Anal. Chem. 2010, 29, 1050–1063.
6.
Oliveira, T.M.B.F.; Ribeiro, F.W.P.; Souza, C. P.; et al. Current overview and perspectives on carbon-based (bio)sensors for carbamate pesticides electroanalysis. Trends Anal. Chem. 2020, 124, 115779.
7.
Kim, K.; Tsay, O.G.; Atwood, D.A.; et al. Destruction and detection of chemical warfare agents. Chem. Rev. 2011, 111, 5345–5403.
8.
Aragay, G.; Pino, F.; Merkoçi, A. Nanomaterials for sensing and destroying pesticides. Chem. Rev. 2012, 112, 5317–5338.
9.
Kundu, M.; Krishnan, P.; Kotnala, R.K.; et al. Recent developments in biosensors to combat agricultural challenges and their future prospects. Trends Food Sci. Technol. 2019, 88, 157–178.
10.
Fang, Y.; Ramasamy, R.P. Current and prospective methods for plant disease detection. Biosensors 2015, 5, 537–561.
11.
Bakker, E.; Qin, Y. Electrochemical sensors. Anal. Chem. 2006, 78, 3965–3984.
12.
Singh, P.; Kamble, G.S.; Ghodake, G.K.; et al. A Newly Emerging Trend of Chitosan-Based Sensing Platform for the Organophosphate Pesticide Detection Using Acetylcholinesterase: A Review. Trends Food Sci. Technol. 2019, 85, 78–91.
13.
Sgobbi, L.F.; Machado, S.A.S. Functionalized polyacrylamide as an acetylcholinesterase-inspired biomimetic device for electrochemical sensing of organophosphorus pesticides. Biosens. Bioelectron. 2018, 100, 290–297.
14.
Ristori, C.; Del Carlo, C.; Martini, M.; et al. Potentiometric detection of pesticides in water samples. Anal. Chim. Acta 1996, 325, 151–160.
15.
Cabral, M.F.; Sgobbi, L.F.; Kataoka, E.M.; et al. On the behavior of acetylcholinesterase immobilized on carbon nanotubes in the presence of inhibitors. Colloids Surf. B 2013, 111, 30–35.
16.
Sgobbi, L.F.; Zibordi-Besse, L.; Rodrigues, B.V.M.; et al. Polyhydroxamicalkanoate as a bioinspired acetylcholinesterase-based catalyst for acetylthiocholine hydrolysis and organophosphorus dephosphorylation: Experimental studies and theoretical insights. Catal. Sci. Technol. 2017, 7, 3388–3398.
17.
Teixeira, S.C.; Gomes, N.O.; Calegaro, M.L.; et al. Sustainable plant-wearable sensors for on-site, rapid decentralized detection of pesticides toward precision agriculture and food safety. Biomater. Adv. 2023, 155, 213676–213686.
18.
Caetano, J.; Machado, S.A.S. Determination of carbaryl in tomato in natura using an amperometric biosensor based on the inhibition of acetylcholinesterase activity. Sens. Actuators B 2008, 129, 40–46.
19.
Patel, H.; Rawtani, D.; Agrawal, Y.K. A newly emerging trend of chitosan-based sensing platform for the organophosphate pesticide detection using acetylcholinesterase—A review. Trends Food Sci. Technol. 2019, 85, 78–91.
20.
Periasamy, A.P.; Umasankar, Y.; Chen, S.-M. Nanomaterials–acetylcholinesterase enzyme matrices for organophosphorus pesticides electrochemical sensors: A review. Sensors 2009, 9, 4034–4055.
21.
Zhu, C.; Yang, G.; Li, H.; et al. Electrochemical sensors and biosensors based on nanomaterials and nanostructures. Anal. Chem. 2015, 87, 230–249.
22.
Wang, J. Nanomaterial-based electrochemical biosensors. Analyst 2005, 130, 421–426.
23.
Pumera, M.; Sanchez, S.; Ichinose, I.; et al. Electrochemical nanobiosensors. Sens. Actuators B 2007, 123, 1195–1205.
24.
Du, D.; Chen, S.; Cai, J.; et al. Immobilization of acetylcholinesterase on gold nanoparticles embedded in sol–gel film for amperometric detection of organophosphorous insecticide. Biosens. Bioelectron. 2007, 23, 130–134.
25.
Castro, F.L.; Castro, A.C.H.; Silva, M.R.A.; et al. Electrochemical detection of SARS-CoV-2 in saliva using ZnO nanorods functionalized with gold-conjugated anti-RBD antibodies. ChemNanoMat 2025, 12, e202500411.
26.
Sabaine, A.E.; Castro, A.C.H.; Mancini, R.S.N.; et al. Peptide-based biosensors for variant-specific detection of SARS-CoV-2 antibodies. Mater. Adv. 2025, 6, 7090–7103.
27.
Nicoliche, C.Y.N.; Pascon, A.M.; Bezerra, Í.R. S.; et al. In situ nanocoating on porous pyrolyzed paper enables antibiofouling and sensitive electrochemical analyses in biological fluids. ACS Appl. Mater. Interfaces 2022, 14, 2522–2533.
28.
Liu, G.; Wang, J.; Barry, R.; et al. Nanoparticle-based electrochemical immunosensor for the detection of phosphorylated acetylcholinesterase: An exposure biomarker of organophosphate pesticides and nerve agents. Chem. Eur. J. 2008, 14, 9951–9959.
29.
Du, D.; Ding, J.; Cai, J.; et al. Electrochemical thiocholine inhibition sensor based on biocatalytic growth of Au nanoparticles using chitosan as template. Sens. Actuators B 2007, 127, 317–322.
30.
Du, D.; Chen, S.; Song, D.; et al. Development of acetylcholinesterase biosensor based on CdTe quantum dots/gold nanoparticles modified chitosan microspheres interface. Biosens. Bioelectron. 2008, 24, 475–479.
31.
Gong, J.; Wang, L.; Zhang, L. Electrochemical biosensing of methyl parathion pesticide based on acetylcholinesterase immobilized onto Au–polypyrrole interlaced network-like nanocomposite. Biosens. Bioelectron. 2009, 24, 2285–2288.
32.
Alves, W.A.; Pelin, J.N.B.D.; Edwards-Gayle, C.J.C.; et al. Self-assembled gold nanoparticles and amphiphile peptides: A colorimetric probe for copper(II) ion detection. Dalton Trans. 2020, 49, 16226–16237.
33.
Gerbelli, B.B.; Vassilaides, S.V.; Rojas, J.E.U.; et al. Hierarchical self-assembly of peptides and its applications in bionanotechnology. Macromol. Chem. Phys. 2019, 220, 1900085.
34.
Pelin, J.N.B.D.; Gatto, E.; Venanzi, M.; et al. Hybrid conjugates formed between gold nanoparticles and an amyloidogenic diphenylalanine-cysteine peptide. ChemistrySelect 2018, 3, 6756–6765.
35.
Karpel, R.L.; Liberato, M.S.; Campeiro, J.D.; et al. Design and characterization of crotamine-functionalized gold nanoparticles. Colloids Surf. B 2018, 163, 1–8.
36.
Wang, S.S. p-Alkoxybenzyl alcohol resin and p-alkoxybenzyloxycarbonylhydrazide resin for solid-phase synthesis of protected peptide fragments. J. Am. Chem. Soc. 1973, 95, 1328–1333.
37.
Turkevich, J.; Stevenson, P.C.; Hillier, J. A study of the nucleation and growth processes in the synthesis of colloidal gold. Discuss. Faraday Soc. 1951, 11, 55–75.
38.
Laviron, E. General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems. J. Electroanal. Chem. 1979, 101, 19–28.
39.
Wessler, I.; Michel-Schmidt, R.; Kirkpatrick, C.J. pH-dependent hydrolysis of acetylcholine: Consequences for non-neuronal acetylcholine. Int. Immunopharmacol. 2015, 29, 27–30.
40.
Štěpánek, P. Data analysis in dynamic light scattering. In Dynamic Light Scattering; Oxford University Press: Oxford, UK, 2023.
41.
Hunter, R.J. Zeta Potential in Colloid Science: Principles and Applications; Academic Press: London, UK, 1981.
42.
Hackley, V.A.; Premachandran, R.S.; Malghan, S.G.; et al. A standard reference material for the measurement of particle mobility by electrophoretic light scattering. Colloids Surf. A 1995, 98, 195–203.
43.
Carone, A.; Emilsson, S.; Mariani, P.; et al. Gold nanoparticle shape dependence of colloidal stability domains. Nanoscale Adv. 2023, 5, 2017–2026.
44.
Väli, R.; Jänes, A.; Lust, E. Alkali-metal insertion processes on nanospheric hard carbon electrodes: An electrochemical impedance spectroscopy study. J. Electrochem. Soc. 2017, 164, E3429–E3436.
45.
Phongphut, A.; Chayasombat, B.; Cass, A.E.; et al. Biosensors based on acetylcholinesterase immobilized on clay–gold nanocomposites for the discrimination of chlorpyrifos and carbaryl. ACS Omega 2022, 7, 39848–39859.
46.
Nunes, E.W.; Silva, M.K.; Rascón, J.; et al. Acetylcholinesterase biosensor based on functionalized renewable carbon platform for detection of carbaryl in food. Biosensors 2022, 12, 486.
47.
Pohanka, M. Electrochemical biosensors based on acetylcholinesterase and butyrylcholinesterase: A review. Int. J. Electrochem. Sci. 2016, 11, 7440–7452.
48.
Tun, W.S.T.; Saenchoopa, A.; Daduang, S.; et al. Electrochemical biosensor based on cellulose nanofibers/graphene oxide and acetylcholinesterase for the detection of chlorpyrifos pesticide in water and fruit juice. RSC Adv. 2023, 13, 9603–9614.
49.
Wang, B.; Li, Y.; Hu, H.; et al. Acetylcholinesterase electrochemical biosensors with graphene-transition metal carbides nanocomposites modified for detection of organophosphate pesticides. PLoS ONE 2020, 15, e0231981.
50.
Hart, J.P.; Hartley, I.C. Voltammetric and amperometric studies of thiocholine at a screen-printed carbon electrode chemically modified with cobalt phthalocyanine: Studies towards a pesticide sensor. Analyst 1994, 119, 259–263.
51.
Oliveira, A.C.; Mascaro, L.H. Evaluation of acetylcholinesterase biosensor based on carbon nanotube paste in the determination of chlorphenvinphos. Int. J. Anal. Chem. 2011, 2011, 974216.
52.
Sucheta, A.; Cammack, R.; Weiner, J.; et al. Reversible electrochemistry of fumarate reductase immobilized on an electrode surface: Direct voltammetric observations of redox centers and their participation in rapid catalytic electron transport. Biochemistry 1993, 32, 5455–5465.
53.
Liu, Q.; Fei, A.; Huan, J.; et al. Effective amperometric biosensor for carbaryl detection based on covalent immobilization of acetylcholinesterase on multiwall carbon nanotubes/graphene oxide nanoribbons nanostructure. J. Electroanal. Chem. 2015, 740, 8–13.
54.
Badawy, M.E.I.; El-Aswad, A.F. Bioactive paper sensor based on acetylcholinesterase for the rapid detection of organophosphate and carbamate pesticides. Int. J. Anal. Chem. 2014, 2014, 536823.
55.
Timur, S.; Telefoncu, A. Acetylcholinesterase (AChE) electrodes based on gelatin and chitosan matrices for pesticide detection. Artif. Cells Blood Substit. Biotechnol. 2004, 32, 427–442.
56.
Zhang, Y.; Liu, H.; Yang, Z.; et al. An acetylcholinesterase inhibition biosensor based on a reduced graphene oxide/silver nanocluster/chitosan nanocomposite for detection of organophosphorus pesticides. Anal. Methods 2015, 7, 6213–6219.
57.
Gong, J.; Liu, T.; Song, D.; et al. One-step fabrication of three-dimensional porous calcium carbonate–chitosan composite film as the immobilization matrix of acetylcholinesterase and its biosensing on pesticide. Electrochem. Commun. 2009, 11, 1873–1876.
58.
Cui, H.-F.; Wu, W.-W.; Li, M.-M.; et al. A highly stable acetylcholinesterase biosensor based on chitosan–TiO₂–graphene nanocomposites for detection of organophosphate pesticides. Biosens. Bioelectron. 2018, 99, 223–229.
59.
Song, D.; Li, Y.; Lu, X.; et al. Palladium–copper nanowires-based biosensor for the ultrasensitive detection of organophosphate pesticides. Anal. Chim. Acta 2017, 982, 168–175.
60.
Zhao, H.; Ji, X.; Wang, B.; et al. An ultra-sensitive acetylcholinesterase biosensor based on reduced graphene oxide–Au nanoparticles–β-cyclodextrin/Prussian blue–chitosan nanocomposites for organophosphorus pesticides detection. Biosens. Bioelectron. 2015, 65, 23–30.
61.
Nesakumar, N.; Ramachandra, B.L.; Sethuraman, S.; et al. Evaluation of inhibition efficiency for the detection of captan, 2,3,7,8-tetrachlorodibenzodioxin, pentachlorophenol, and carbosulfan in water: An electrochemical approach. Bull. Environ. Contam. Toxicol. 2016, 96, 217–223.
62.
Guo, Y.; Sun, X.; Liu, X.; et al. A miniaturized portable instrument for rapid determination of pesticide residues in vegetables and fruits. IEEE Sens. J. 2015, 15, 4046–4052.
63.
Du, D.; Huang, X.; Cai, J.; et al. Comparison of pesticide sensitivity by electrochemical test based on acetylcholinesterase biosensor. Biosens. Bioelectron. 2007, 23, 285–289.
64.
Du, D.; Ding, J.; Tao, Y.; et al. Application of chemisorption/desorption process of thiocholine for pesticide detection based on acetylcholinesterase biosensor. Sens. Actuators B 2008, 134, 908–912.
65.
Sun, X.; Wang, X. Acetylcholinesterase biosensor based on Prussian blue–modified electrode for detecting organophosphorous pesticides. Biosens. Bioelectron. 2010, 25, 2611–2614.
66.
Yan, J.; Guan, H.; Yu, J.; et al. Acetylcholinesterase biosensor based on assembly of multiwall carbon nanotubes onto liposome bioreactors for detection of organophosphate pesticides. Pestic. Biochem. Physiol. 2013, 105, 197–202.
67.
Guan, H.; Zhang, F.; Yu, J.; et al. Novel acetylcholinesterase biosensors based on liposome bioreactors–chitosan nanocomposite film for detection of organophosphate pesticides. Food Res. Int. 2012, 49, 15–21.
68.
Sun, X.; Li, Q.; Wang, X. AChE biosensor based on aniline–MWNTs modified electrode for the detection of pesticides. In Proceedings of the 2011 IEEE/ICME International Conference on Complex Medical Engineering, Harbin, China, 22–25 May 2011; pp 441–444.
69.
Zhao, Y.; Zhang, S.P.; Ma, J.; et al. Determination of carbamate pesticide methomyl using acetylcholinesterase/MWNTs–chitosan modified glassy carbon electrode. Int. J. Electrochem. Sci. 2012, 7, 8856–8867.
70.
Dong, J.; Zhao, H.; Qiao, F.; et al. Quantum dot immobilized acetylcholinesterase for the determination of organophosphate pesticides using graphene–chitosan nanocomposite modified electrode. Anal. Methods 2013, 5, 2866–2872.
71.
Du, D.; Wang, M.; Cai, J.; et all. One-step synthesis of multiwalled carbon nanotubes–gold nanocomposites for fabricating amperometric acetylcholinesterase biosensor. Sens. Actuators B 2010, 143, 524–529.
72.
Zhai, C.; Sun, X.; Zhao, W.; et al. Acetylcholinesterase biosensor based on chitosan/Prussian blue/multiwall carbon nanotubes/hollow gold nanospheres nanocomposite film by one-step electrodeposition. Biosens. Bioelectron. 2013, 42, 124–130.
73.
Kandimalla, V.B.; Ju, H. Binding of acetylcholinesterase to multiwall carbon nanotube–cross-linked chitosan composite for flow-injection amperometric detection of an organophosphorous insecticide. Chem. Eur. J. 2006, 12, 1074–1080.
74.
El-Moghazy, A.Y.; Soliman, E.A.; Ibrahim, H.Z.; et al. Biosensor based on electrospun blended chitosan–poly(vinyl alcohol) nanofibrous enzymatically sensitized membranes for pirimiphos-methyl detection in olive oil. Talanta 2016, 155, 258–264.
75.
Talarico, D.; Arduini, F.; Amine, A.; et al. Screen-printed electrode modified with carbon black and chitosan: A novel platform for acetylcholinesterase biosensor development. Anal. Bioanal. Chem. 2016, 408, 7299–7309.
76.
Du, D.; Ding, J.; Cai, J.; et al. One-step electrochemically deposited interface of chitosan–gold nanoparticles for acetylcholinesterase biosensor design. J. Electroanal. Chem. 2007, 605, 53–60.
77.
Yang, Y.; Guo, M.; Yang, M.; et al. Determination of pesticides in vegetable samples using an acetylcholinesterase biosensor based on nanoparticles ZrO₂/chitosan composite film. Int. J. Environ. Anal. Chem. 2005, 85, 163–175.
78.
Liu, T.; Su, H.; Qu, X.; et al. Acetylcholinesterase biosensor based on 3-carboxyphenylboronic acid/reduced graphene oxide–gold nanocomposites modified electrode for amperometric detection of organophosphorus and carbamate pesticides. Sens. Actuators B 2011, 160, 1255–1261.

Scilight Press

Author Information

Abstract

Graphical Abstract

Keywords

References

About Scilight

Journals

Publishing Policies

Contact Us