Microchimica Acta

, Volume 182, Issue 3–4, pp 643–651 | Cite as

Screen-printed biosensor modified with carbon black nanoparticles for the determination of paraoxon based on the inhibition of butyrylcholinesterase

  • Fabiana Arduini
  • Matteo Forchielli
  • Aziz Amine
  • Daniela Neagu
  • Ilaria Cacciotti
  • Francesca Nanni
  • Danila Moscone
  • Giuseppe Palleschi
Original Paper


We have developed a screen-printed electrochemical electrode (SPE) for paraoxon based on its inhibitory effect on the enzyme butyrylcholinesterase (BChE). The electrode was first modified by drop casting with a dispersion of carbon black nanoparticles (CBNPs) in a dimethylformamide-water mixture, and BChE was then immobilized on the surface by cross-linking. The resulting biosensor was exposed to standard solutions of paraoxon, and the enzymatic hydrolysis of butyrylthiocholine over time was determined measuring the enzymatic product thiocholine at a working voltage of +300 mV. The enzyme inhibition is linearly related to the concentration of paraoxon up to 30 μg L−1, and the detection limit is 5 μg L−1. The biosensor is stable for up to 78 days of storage at room temperature under dry conditions. It was applied to determined paraoxon in spiked waste water samples. The results underpin the potential of the use of CBNPs in electrochemical biosensors and also demonstrate that they represent a viable alternative to other carbon nanomaterials such as carbon nanotubes or graphene, and with the advantage of being very affordable.

Graphical Abstract

A screen-printed electrode (SPE) for paraoxon detection at ppb levels based on its inhibitory effect on the enzyme butyrylcholinesterase was developed. The measurement is characterised by absence of fouling problem due to the use of carbon black nanoparticles.


Organophosphate Butyrylcholinesterase Screen-printed electrode Carbon black nanoparticles Inhibition 



This work was supported by National Industria 2015 (MI01_00223) ACQUA-SENSE project and Marie Curie FP7-PEOPLE-2011-IRSES, 294901 “Peptide Nanosensors”. The authors thank Prof. F. Cataldo (Actinium Chemical Research srl) for the CBNPs samples, Tover Italia s.r.l. (Rome) and BASF Italia Divisione Catalizzatori (Rome) for the waste water samples.


  1. 1.
    Bergh C, Torgrip R, Ostman C (2010) Simultaneous selective detection of organophosphate and phthalate esters using gas chromatography with positive ion chemical ionization tandem mass spectrometry and its application to indoor air and dust. Rapid Commun Mass Spectrom 24:2859–2867CrossRefGoogle Scholar
  2. 2.
    Freitas Ventura F, Oliveira J, Pedreira Filho WDR, Gerardo Ribeiro M (2012) GC-MS quantification of organophosphorous pesticides extracted from XAD-2 sorbent tube and foam patch matrices. Anal Methods 4:3666–3673CrossRefGoogle Scholar
  3. 3.
    Rosenfeld CA, Sultatos LG (2006) Concentration-dependent kinetics of acetylcholinesterase inhibition by the organophosphate paraoxon. Toxicol Sci 90:460–469CrossRefGoogle Scholar
  4. 4.
    Andreescu S, Marty JL (2006) Twenty years research in cholinesterase biosensors: From basic research to practical applications. Biomol Eng 23:1–15CrossRefGoogle Scholar
  5. 5.
    Arduini F, Amine A, Moscone D, Palleschi G (2010) Biosensors based on cholinesterase inhibition for insecticides, nerve agents and aflatoxin B1 detection (review). Microchim Acta 170:193–214CrossRefGoogle Scholar
  6. 6.
    Taleat Z, Khoshroo A, Mazloum-Ardakani M (2014) Screen-printed electrodes for biosensing: a review (2008–2013) Microchim Acta, in press, doi:  10.1007/s00604-014-1181-1
  7. 7.
    Fennouh S, Casimiri V, Burstein C (1997) Increased paraoxon detection with solvents using acetylcholinesterase inactivation measured with a choline oxidase biosensor. Biosens Bioelectron 12:97–104CrossRefGoogle Scholar
  8. 8.
    Cremisini C, Di Sario S, Mela J, Pilloton R, Palleschi G (1995) Evaluation of the use of free and immobilised acetylcholinesterase for paraoxon detection with an amperometric choline oxidase based biosensor. Anal Chim Acta 311:273–280CrossRefGoogle Scholar
  9. 9.
    Hart JP, Hartley IC (1994) Voltammetric and amperometric studies of thiocholine at screen-printed carbon electrode chemically modified with cobalt phthalocyanine: studies towards a pesticide sensor. Analyst 119:259–263CrossRefGoogle Scholar
  10. 10.
    Ricci F, Arduini F, Amine A, Moscone D, Palleschi G (2004) Characterisation of Prussian blue modified screen-printed electrodes for thiol detection. J Electroanal Chem 563:229–237CrossRefGoogle Scholar
  11. 11.
    Montesinos T, Pérez-Munguia S, Valdez F, Marty JL (2001) Disposable cholinesterase biosensor for the detection of pesticides in water miscible organic solvents. Anal Chim Acta 431:231–237CrossRefGoogle Scholar
  12. 12.
    Arduini F, Cassisi A, Amine A, Ricci F, Moscone D, Palleschi G (2009) Electrocatalytic oxidation of thiocholine at chemically modified cobalt hexacyanoferrate screen-printed electrodes. J Electroanal Chem 626:66–74CrossRefGoogle Scholar
  13. 13.
    Arduini F, Guidone S, Amine A, Palleschi G, Moscone D (2013) Acetylcholinesterase biosensor based on self-assembled monolayer-modified gold-screen printed electrodes for organophosphorus insecticide detection. Sens Actuat B 179:201–208CrossRefGoogle Scholar
  14. 14.
    Joshi KA, Tang J, Haddon R, Wang J, Chen W, Mulchandani A (2005) A disposable biosensor for organophosphorus nerve agents based on carbon nanotubes modified thick film strip electrode. Electroanal 17:54–58CrossRefGoogle Scholar
  15. 15.
    Arduini F, Amine A, Majorani C, Di Giorgio F, De Felicis D, Cataldo C, Moscone D, Palleschi G (2010) High performance electrochemical sensor based on modified screen-printed electrodes with cost-effective dispersion of nanostructured carbon black. Electrochem Commun 12:346–350CrossRefGoogle Scholar
  16. 16.
    Arduini F, Majorani C, Amine A, Moscone D, Palleschi G (2011) Hg2+ detection by measuring thiol groups with a highly sensitive screen-printed electrode modified with a nanostructured carbon black film. Electrochim Acta 56:4209–4215CrossRefGoogle Scholar
  17. 17.
    Arduini F, Di Nardo F, Amine A, Micheli L, Palleschi G, Moscone D (2012) Carbon black-modified screen-printed electrodes as electroanalytical tools. Electroanal 24:743–751CrossRefGoogle Scholar
  18. 18.
    Suprun E, Arduini F, Moscone D, Palleschi G, Shumyantseva VV, Archakov AI (2012) Direct electrochemistry of Heme proteins on electrodes modified with didodecyldimethyl ammonium bromide and carbon black. Electroanal 24:1923–1931CrossRefGoogle Scholar
  19. 19.
    Lo TWB, Aldousa L, Compton RG (2012) The use of nano-carbon as an alternative to multi-walled carbon nanotubes in modified electrodes for adsorptive stripping voltammetry. Sens Actuat B 162:361–368CrossRefGoogle Scholar
  20. 20.
    Ellman GL (1958) A colorimetric method for determining low concentrations of mercaptans. Arch Biochem Biophys 74:443–445CrossRefGoogle Scholar
  21. 21.
    Liu G, Riechers SL, Mellen MC, Lin Y (2005) Sensitive electrochemical detection of enzymatically generated thiocholine at carbon nanotube modified glassy carbon electrode. Electrochem Commun 7(11):1163–1169CrossRefGoogle Scholar
  22. 22.
    Liu G, Lin Y (2006) Biosensor based on self-assembling acetylcholinesterase on carbon nanotubes for flow injection/amperometric detection of organophosphate pesticides and nerve agents. Anal Chem 78(3):835–843CrossRefGoogle Scholar
  23. 23.
    Li Y, Bai Y, Han G, Li M (2013) Porous-reduced graphene oxide for fabricating an amperometric acetylcholinesterase biosensor. Sens Actuat B 185:706–712CrossRefGoogle Scholar
  24. 24.
    Wang K, Liu Q, Dai L, Yan J, Ju C, Qiu B, Wu X (2011) A highly sensitive and rapid organophosphate biosensor based on enhancement of CdS–decorated graphene nanocomposite. Anal Chim Acta 695:84–88CrossRefGoogle Scholar
  25. 25.
    Liu T, Su H, Qu X, Ju P, Cui L, Ai S (2011) Acetylcholinesterase biosensor based on 3-carboxyphenylboronic acid/reduced graphene oxide–gold nanocomposites modified electrode for amperometric detection of organophosphorus and carbamate pesticides. Sens Actuat B 160:1255–126CrossRefGoogle Scholar
  26. 26.
    Du D, Chen S, Song D, Li H, Chen X (2008) Development of acetylcholinesterase biosensor based on CdTe quantum dots/gold nanoparticles modified chitosan microspheres interface. Biosens Bioelectron 24:475–479CrossRefGoogle Scholar
  27. 27.
    Zhang H, Li ZF, Snyder A, Xie J, Stanciu LA (2014) Functionalized graphene oxide for the fabrication of paraoxon biosensors. Anal Chim Acta 827:86–94CrossRefGoogle Scholar
  28. 28.
    Ganesana M, Istarnboulie G, Marty JL, Noguer T, Andreescu S (2011) Site-specific immobilization of a (His) 6-tagged acetylcholinesterase on nickel nanoparticles for highly sensitive toxicity biosensors. Biosens Bioelectron 30:43–48CrossRefGoogle Scholar
  29. 29.
    Ivanov AN, Younusov RR, Evtugyn GA, Arduini F, Moscone D, Palleschi G (2011) Acetylcholinesterase biosensor based on single-walled carbon nanotubes-Co phtalocyanine for organophosphorus pesticides detection. Talanta 85:216–221CrossRefGoogle Scholar
  30. 30.
    Pohanka M, Jun D, Kuca K (2008) Amperometric biosensor for real time assay of organophosphates. Sensors 8:5303–5312CrossRefGoogle Scholar
  31. 31.
    Zamfir LG, Rotariu L, Bala C (2011) A novel, sensitive, reusable and low potential acetylcholinesterase biosensor for chlorpyrifos based on 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotubes gel. Biosens Bioelectron 26:3692–3695CrossRefGoogle Scholar
  32. 32.
    Arduini F, Palleschi G (2012) Disposable Electrochemical Biosensor Based on Cholinesterase Inhibition with Improved Shelf-Life and Working Stability for Nerve Agent Detection. In: Nikolelis DP (ed) NATO science for peace and security series A: chemistry and biology portable chemical sensors weapons against bioterrorism. Springer, The Netherlands, pp 261–278Google Scholar
  33. 33.
    Arduini F, Neagu D, Dall’Oglio S, Moscone D, Palleschi G (2012) Towards a portable prototype based on electrochemical cholinesterase biosensor to be assembled to soldier overall for nerve agent detection. Electroanalysis 24:581–590CrossRefGoogle Scholar
  34. 34.
    Arduini F, Ricci F, Tuta CS, Moscone D, Amine A, Palleschi G (2006) Detection of carbamic and organophosphorus pesticides in water samples using a cholinesterase biosensor based on Prussian Blue-modified screen-printed electrode. Anal Chim Acta 580:155–162CrossRefGoogle Scholar
  35. 35.
    Lee JH, Park JY, Min K, Cha HJ, Choi SS, Yoo YJ (2010) A novel organophosphorus hydrolase-based biosensor using mesoporous carbons and carbon black for the detection of organophosphate nerve agents. Biosens Bioelectron 25:1566–1570CrossRefGoogle Scholar
  36. 36.
    Tang X, Zhang T, Liang B, Han D, Zeng L, Zheng C, Li T, Wei M, Liu A (2014) A. Sensitive electrochemical microbial biosensor for p-nitrophenylorganophosphates based on electrode modified with cell surface-displayed organophosphorus hydrolase and ordered mesopore carbons. Biosens Bioelectron 60:137–142CrossRefGoogle Scholar
  37. 37.
    Crew A, Lonsdale D, Byrd N, Pittson R, Hart JP (2011) A screen-printed, amperometric biosensor array incorporated into a novel automated system for the simultaneous determination of organophosphate pesticides. Biosens Bioelectron 26:2847–2851CrossRefGoogle Scholar
  38. 38.
    Tan HY, Loke WK, Nguyen NT, Tan SN, Tay NB, Wang W, Ng SH (2014) Lab-on-a-chip for rapid electrochemical detection of nerve agent Sarin. Biomed Microdevices 16(2):269–275CrossRefGoogle Scholar
  39. 39.
    DL 152/2006, 3 April 2006 “Norme in materia ambientale”,

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Fabiana Arduini
    • 1
  • Matteo Forchielli
    • 1
  • Aziz Amine
    • 2
  • Daniela Neagu
    • 1
  • Ilaria Cacciotti
    • 3
  • Francesca Nanni
    • 4
  • Danila Moscone
    • 1
  • Giuseppe Palleschi
    • 1
  1. 1.Dipartimento di Scienze e Tecnologie ChimicheUniversità di Roma Tor Vergata, Via della Ricerca ScientificaRomeItaly
  2. 2.Faculté de Sciences et Techniques Laboratoire Génie des Procédés et EnvironnementUniversité Hassan II-MohammediaMohammadiaMorocco
  3. 3.Università degli Studi di Roma “Niccolò Cusano”, UdR INSTMRomeItaly
  4. 4.Dipartimento di Ingegneria dell’ImpresaUniversità di Roma Tor Vergata, UdR INSTM Roma-Tor VergataRomeItaly

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