Acetylcholinesterase sensor based on PANi/rGO film electrochemically grown on screen-Printed electrodes

Cite this paper: Vietnam J. Chem., 2021, 59(2), 253-262 Article 
DOI: 10.1002/vjch.202000158 
253 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH 
Acetylcholinesterase sensor based on PANi/rGO film electrochemically 
grown on screen-printed electrodes 
Ly Cong Thanh
1
, Dau Thi Ngoc Nga
2
, Nguyen Viet Bao Lam
3
, Pham Do Chung
3
, Le Thi Thanh Nhi
4
, 
Le Hoang Sinh
4
, Vu Thi Thu
2*
,
Tran Dai Lam
5* 
1
Hanoi University of Pharmacy (HUP), 15-17 Le Thanh Tong, Hoan Kiem, Hanoi 10000, Viet Nam 
2
University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology 
(VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 
3
Hanoi National University of Education (HNUE), 134-136 Xuan Thuy, Cau Giay, Hanoi 10000, Viet Nam 
4
Duy Tan University (DTU), 03 Quang Trung, Da Nang 50000, Viet Nam 
5
Institute of Tropical Technology (ITT), VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 
Submitted September 11, 2020; Accepted February 24, 2021 
Abstract 
In this work, the polyaniline/reduced graphene oxide (PANi/rGO) bilayer was directly electrodeposited on carbon 
screen-printed electrodes (SPE). Some details in growth of PANi/rGO bilayer were revealed from cyclic 
voltammograms and X-ray photoelectron spectra. The growth of stacked rGO film at high compactness on the electrode 
surface is mainly accompanied with reduction of epoxy functional groups at basal planes of graphitic flakes. The as-
grown rGO layer with abundent hydroxyl functional groups at basal planes is preferable to attract intrinsic fibrillar-like 
PANi polymer chains in protonated aqueous media. The as-prepared PANi/rGO hybrid bilayer has shown good 
conductivity, high porosity, good adhesion to biomolecules, and fast electron transfer rate (increased by 3.8 times). 
Herein, PANi/rGO film has been further utilized to develop disposable acetylcholinesterase sensors able to detect 
acetylthiocholine (ATCh) with apparent Michaelis - Menten constant of 0.728 mM. These sensors provide a very 
promising technical solution for in-situ monitoring acetylthiocholine level in patients with neuro-diseases and 
determination of neuro-toxins such as sarin and pesticides. 
Keywords. Reduced graphene oxide (rGO), polyaniline (PANi), acetylcholinesterase (AChE), screen-printed 
electrodes (SPE), neuro-diseases, electrodeposition. 
1. INTRODUCTION 
Hybrid films which combined biocompatible 
polymers and highly conductive inorganic 
nanomaterials have recently gained many attentions 
in sensing and electronic applications. Among well-
known conducting nanomaterials, graphene and its 
derivatives with extraordinary conductivity, 
mechanical stability and flexibility are the best 
candidates that meet many critical requirements of 
electrochemical sensing systems.
[1]
 Especially, 
reduced graphene oxide (rGO) is the most frequently 
used since it provides many behaviors similar with 
graphene and can be easily produced at large 
scale
[2,3]
 through solution-based approaches and 
combined with other materials in composites.
[4,5]
Meanwhile, polyaniline (PANi) with good 
conductivity, high porosity, and good adhesion to 
biomolecules (i.e. enzymes) is often utilized in 
electrochemical biosensors. Interestingly, PANi has 
three different chemical states that can be tuned 
electrochemically
[6,7]
 and sensitive to 
protonation/deprotonation process.
[8]
 Also, the 
presence of amino groups in polymer chains of 
PANi make it becomes one favorable transducing 
platform to immobilize enzymes. Probably, the 
hybrid structures based on PANi and carbonaceous 
materials should have inherited the mentioned 
benefits of these two materials. 
Several research groups have demonstrated 
potential applications of hybrid films based on 
carbonaceous nanomaterials with PANi. Depending 
on the purpose of the application, these hybrid films 
were grown either in composite structure or bilayer 
architecture. In the beginning, composite films based 
on graphene derivatives and PANi were mainly 
Vietnam Journal of Chemistry Vu Thi Thu et al. 
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 254 
utilized for developing high-performance 
supercapacitors in flexible energy storage devices.
[9-
11]
 These hybrid composites also show high anti-
corrosion behavior.
[12]
 Recently, the layer-by-layer 
structure of hybrid films made of conducting 
polymers and carbonaceous materials has drawn 
more attentions. The assembly of the two distinct 
materials in two separated layers allows better 
control in their thickness and homogeneity. The use 
of graphitic material as one supporting layer 
provides the solution to overcome insulating nature 
and structural shrinkage of PANi in dedoping 
states.
[13,14]
 Moreover, the addition of soft PANi 
material make carbonaceous materials become less 
rigid and more biocompatible. For instance, the 
PANi ad-layer electrodeposited on graphitic 
electrodes has been shown to improve voltammetric 
signals during analysis of redox probes.
[15]
PANi/graphene bilayer with good conductivity and 
fast electron transfer has been shown to be profitable 
in electrochemical immunosensors for tracing neuro-
toxins.
[16]
 PANi/rGO bilayer was utilized as one pH-
sensitive membrane to sense protons released from 
gene amplification process.
[17]
 Some suggestions on 
structure of PANi/rGO bilayer were previously 
provided but the details on growth mechanism of 
this hybrid bilayer is still unclear until now. 
Many neurodegenerative diseases (i.e, 
Alzheimer’s disease and Parkinson’s disease) are 
associated with the degeneration of the cholinergic 
system that is caused by abnormal AChE activity. 
Therefore, it is essential to develop realiable tools 
for monitoring the activities of AChE en ... ects of reduction degree on 
concentration of OFGs (i.e. hydroxyl groups) on 
morphology and charge transfer kinetics of hybrid 
films based on rGO and several conducting 
polymers will be studied. 
Table 3: Comparisons between AChE 
electrochemical sensors 
Configuration 
Linear 
range 
Dection 
limit 
(µM) 
Km 
(mM) 
Ref. 
Pd@Au/AChE 
4-124 
µM 
- 0.19 [20] 
GCE/rGO/CS@
TiO2-CS/AChE 
0.1-9.0 
mM 
- 3.1 [37] 
GCE/Pd@AuN
Rs/AChE-
CS/Nafion 
2-272 
µM - 0.207 [38] 
Graphite 
electrode/poly(F
BThF)/MNPs/A
ChE 
0.125-
2.6 
mM 
6.66 0.731 [39] 
GCE/PDDA/PS
S/AChE 
1 µM-
10 mM 
- 2.16 [40] 
GCE/Gr-
MNPs/AChE 
12.5-
112.5 
µM 
8.35 - [41] 
SPE/rGO/PANi/
AChE 
0.192-
1.094 
mM 
17.5 0.728 
This 
work 
Note: CS = chitosan, NRs = nanorods; 
FBThF = 4,7-di(furan-2-yl)benzo[c][1,2,5]thiadiazole; 
MNPs = magnetic nanoparticles; 
PDDA = poly(diallyldimethylammonium chloride), PSS 
= polystyrene sulfonate. 
Declaration of interest. The authors have no 
financial interests to declare. 
Acknowledgment. This research is funded by 
Vietnam National Foundation for Science and 
Technology Development NAFOSTED (grant 
number 104.03-2018.344 and 103.02-2018.360). 
The authors also express great thanks to our 
colleagues at Hanoi National University of 
Education (Hanoi, Vietnam) for their supports in 
Raman measurements and our colleagues at 
University of Paris-Sarclay (Paris, France) for their 
supports in XPS measurements. 
0.2 0.4 0.6 0.8 1.0 1.2
0
300
600
900
1200
1500
1800
I 
(n
A
)
C (mM)
Vietnam Journal of Chemistry Vu Thi Thu et al. 
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 260 
REFERENCES 
1. Y. Song, Y. Luo, C. Zhu, H. Li, D. Du, Y. Lin. 
Recent advances in electrochemical biosensors based 
on graphene two-dimensional nanomaterials, 
Biosens. Bioelectron., 2016, 76, 195-212. 
2. Z. Luo, Y. Lu, LA. Somers, AT. Charlie Johnson. 
High yeild preparation of macroscopic graphene 
oxide membranes, J. Am. Chem. Soc., 2009, 131, 
898-899. 
3. H. Wang, JT. Robinson, X. Li, H. Dai. Solvothermal 
reduction of chemically exfoliated graphene sheets, J. 
Am. Chem. Soc., 2009, 131, 9910-9911. 
4. TMBF. Oliveira, FWP. Ribeiro, CP. Sousa, GR. 
Salazar-Band, P. Lima-Neto, A.N Correia, S. Morais. 
Current overview and perspectives on carbon-based 
(bio)sensors for carbamate pesticides electroanalysis, 
Trends Anal. Chem., 2020, 124, 115779. 
5. L. Zhang, Z. Liu, Q. Xie, Y. Li, Y. Ying, Y. Fu. Bio-
inspired assembly of reduced graphene oxide by 
fibrin fiber to prepare multi-functional conductive 
bion-nanocomposites as versatile electrochemical 
platforms, Carbon, 2019, 153, 504-512. 
6. AJ. Motheo, JR. Santos Jr, EC. Venancio, LHC. 
Mattoso. Influence of different types of acidic 
dopants on the electrodeposition and properties of 
polyaniline films, Polymer, 1998, 39, 6977-6982. 
7. A. Nautiyal, JE. Cook, X. Zhang. Tunable 
electrochemical performance of polyaniline coating 
via facile ion exchanges, Prog. Org. Coat., 2019, 
136, 105309. 
8. HJ. Nogueira, PD. Mello, M. Mulato. Influence of 
galvanostatic electrodeposition parameters on the 
structure property relationships of polyaniline thin 
films and their use as potentiometric and optical pH 
sensors, Thin Solid Films, 2018, 656, 14-21. 
9. D. Liu, H. Wang, P. Du, W. Wei, Q. Wang, P. Liu. 
Flexible and robust reduced graphene oxide/carbon 
nanoparticles/polyaniline (RGO/CNs/PANI) 
composite films: Excellent candidates as free-
standing electrodes for high-performance 
supercapacitors, Electrochim. Acta, 2018, 259, 161-
169. 
10. A. Aydinli, R. Yuksel, HE. Unalan. Vertically 
aligned carbon nanotube - Polyaniline nanocomposite 
supercapacitor electrodes, Int. J. Hydrog., 2018, 43, 
18617-18625. 
11. KG. Laelabadi, R. Moradian, I. Manouchehri. One-
step fabrication of flexible, cost/time effective, and 
high energy storage reduced graphene oxide@PANi 
supercapacitor, ACS Appl. Energy Mater., 2020, 3, 
5301-5312. 
12. S. Liu, L. Liu, H. Guo, EE. Oguzie, Y. Li, F. Wang. 
Electrochemical polymerization of polyaniline-
reduced graphene oxide composite coating on 5083 
Al alloy: Role of reduced graphene oxide, 
Electrochem. Commun., 2019, 98, 110-114. 
13. M. Zhang, Y. Zhang, J. Yuan, Y. Zhao, L. Yang, Z. 
Dai, J. Tang. High rate capability electrode from a 
ternary composite of nanodiamonds/reduced 
graphene oxide@PANi for electrochemical 
capacitors, Chem. Phys., 2019, 526, 110461. 
14. J. Ma, J. Dai, Y. Duan, J. Zhang, L. Qiang, J. Xue. 
Fabrcation of PANi-TiO2/rGO hybrid composites for 
enhanced photocatalysis of pollutant removal and 
hydrogen production, Renew. Energy, 2020, 156, 
1008-1018. 
15. CS. Camacho, JC. Mesquita, J. Rodrigues. 
Electrodeposition of polyaniline on self-assembled 
monolayers on graphite for the voltammetric 
detection of iron(II), Mater. Chem. Phys., 2016, 184, 
261-268. 
16. Nguyen VC., Nguyen HB., Cao TT., Nguyen VT., 
Nguyen LH., Nguyen TD., Phan NM., Vu TT., Tran 
DL. Electrochemical immunosensor for detection of 
atrazine based on polyaniline/graphene, J. Mater. Sci. 
Technol., 2016, 32, 539-544. 
17. Vu TT., Bui QT., Dau TNN., Ly CT., Le HS., Le 
CT., Tran DL. Reduced graphene oxide-polyaniline 
film as enhanced sensing interface for the detection 
of loop-mediated-isothermal-amplification products 
by open circuit potential measurement, RSC Adv., 
2018, 8, 25361-25367. 
18. P. Dong, Y. Liu, Y. Zhao, W. Wang, M. Pan, Y. Liu, 
X. Liu. Ratiometric fluorescence sensing of copper 
ion and enzyme activity by nanoprobe-medicated 
autocatalytic reaction and catalytic cascade reaction, 
Sens. Actuators B Chem., 2020, 310, 127873. 
19. P. Zhang, C. Fu, Y. Xiao, Q. Zhang, C. Ding. Copper 
(II) complex as a turn on fluorescent sensing platform 
for acetylcholinesterase activity with high sensitiviy, 
Talanta, 2020, 208, 120406. 
20. M. Wang, L. Liu, X. Xie, X. Zhou, Z. Lin, X. Su. 
Single-atom iron containing nanozyme with 
peroxidase-like activity and copper nanoclusters 
based ratio fluorescent strategy for 
acetylcholinesterase activity sensing, Sens. Actuators 
B Chem., 2020, 313, 128023. 
21. S. Kurbanoglu, C. Erkmen, B. Uslu. Frontiers in 
electrochemical enzyme based biosensors for food 
and drug analysis, Trends Anal. Chem., 2020, 124, 
115809. 
22. H. Shimada, Y. Kiyozumi, Y. Koga, Y. Ogata, Y. 
Katsuda, Y. Kitamura, M. Iwatsuki, K. Nishiyama, 
H. Baba, T. Ihara. A novel cholinesterase assay for 
the evaluation of neurotoxin poisoning based on the 
electron-transfer promotion effect of thiocholine on 
an Au electrode, Sens. Actuators B Chem., 2019, 298, 
126893. 
23. X. Lu, L. Tao, Y. Li, H. Huang, F. Gao. A highly 
sensitive electrochemical platform based on the 
bimetallic Pd@Au nanowires network for 
Vietnam Journal of Chemistry Acetylcholinesterase sensor based on 
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 261 
organophosphorus pesticides detection, Sens. 
Actuators B Chem., 2019, 284, 103-109. 
24. QT Hua, N. Ruecha, Y. Hiruta, D. Citterio. 
Disposable electrochemical biosensor based on 
surface-modified screen-printed electrodes for 
organophosphorus pesticide analysis, Anal. Methods, 
2019, 11, 3439-3445. 
25. B. Zou, Y. Chu, J. Xia. Monocrotophos detection 
with a bienzyme biosensor based on ionic liquid 
modified carbon nanotubes, Anal. Bioanal. Chem., 
2019, 411, 2905-2914. 
26. J. Bao, T. Huang, Z. Wang, H. Yang, X. Geng, G. 
Xu, M. Samalo, M. Sakinati, D. Huo, C. Hou. 3D 
graphene/copper oxide nano-flowers based 
acetylcholinesterase biosensor for sensitive detection 
of organophosphate pesticides, Sens. Actuators B 
Chem., 2019, 284, 95-101. 
27. S. Nagabooshanam, AT. John, S. Wadhwa, A. 
Mathur, S. Krishnamurthy, LM. Bharadwaj. Electro-
deposited nano-webbed structures based on 
polyaniline/multiwalled carbon nanotubes for 
enzymatic detection of organophosphates, Food 
Chem., 2020, 323, 126784. 
28. Dau TNN., Vu VH., Cao TT., Nguyen VC., Ly CT., 
Tran DL., Truong Thuan Nguyen Pham, Nguyen TL., 
Benoit Piro, Vu TT. In-situ electrochemically 
deposited Fe3O4 nanoparticles onto graphene 
nanosheets as amperometric amplifier for 
electrochemical biosensing applications, Sens. 
Actuators B Chem., 2019, 283, 52-60. 
29. Vu TT., Dau TNN., Ly CT., Pham DC., Nguyen 
TTN., Pham VT. Aqueous electrodeposition of 
(AuNPs/MWCNT-PEDOT) composite for high-
affinity acetlcholinesterase electrochemical sensors, 
J. Mater. Sci., 2020, 55, 9070-9081. 
30. Le TTN., Le VT., Dao MU., Nguyen QV., Vu TT., 
Nguyen MH., Tran DL., Le HS. Preparation of 
magnetic graphene oxide/chitosan composite beads 
for effective removal of heavy metals and dyes from 
aqueous solutions, Chem. Eng. Commun., 2019, 206, 
1-16. 
31. KN. Kudin, B. Ozbas, HC. Schniepp, RK. 
Prud’homme, IA. Aksay, R. Car. Raman Spectra of 
Graphite Oxide and Functionalized Graphene, Nano 
Letter, 2008, 8, 36-41. 
32. A. Ambrosi, CK. Chua, NM. Latiff, AH. Loo, CHA. 
Wong, AYS. Eng, A. Bonanni, M. Pumera. Graphene 
and its electrochemistry - an update, Chem. Soc. Rev., 
2016, 45, 2458-2492. 
33. DR. Dreyer, S. Park, CW. Bielawski, RS. Ruoff. The 
chemistry of graphene oxide, Chem. Soc. Rev., 2010, 
39, 228-240. 
34. AG. Marrani, A. Motta, R. Schrebler, R. Zanoni, EA. 
Dalchiele. Insights from experiment and theory into 
the electrochemical reduction mechanism of 
graphene oxide, Electrochim. Acta, 2019, 304, 231-
238. 
35. A. Buchsteiner, A. Lerf, J. Pieper. Water dynamics in 
graphite oxide investigated with neutron scattering, J. 
Phys. Chem. B, 2006, 110, 22328-22338. 
36. I. Turyan, D. Mandler. Two-dimensional polyaniline 
thin film electrodeposited on a self-assembled 
monolayer, J. Am. Chem. Soc., 1998, 120, 10733-
10742. 
37. Z. Mandic, L. Duic, F. Kovacicek. The influence of 
counter-ions on nucleation and growth of 
electrochemically synthesized polyaniline film, 
Electrochim. Acta, 1997, 42, 1389-1402. 
38. X. Zhao, Y. You, S. Huang, F. Cheng, P. Chen, H. 
Li, Y. Zhang. Facile construction of reduced 
graphene oxide supported three-dimensional 
polyaniline/WO2.72 nanobelt-flower as a full solar 
spectrum light response catalyst for efficient 
photocatalytic conversion of bromate, Chemosphere, 
2019, 222, 781-788. 
39. R. Arukula, M. Vinothkannan, AR. Kim, DJ. Yoo. 
Cumulative effect of bimetallic alloy, conductive 
polymer and graphene toward electrooxidation of 
methanol: An efficient anode catalyst for direct 
methanol fuel cells, J. Alloys Compd., 2019, 771, 
477-488. 
40. S. Gao, L. Zhang, Y. Qiao, P. Dong, J. Shi, S. Cao. 
Electrodeposition of polyaniline on three-
dimensional graphen hydrogel as a binder-free 
supercapacitor electrode with high power and energy 
densities, RSC Adv., 2016, 6, 58854-58861. 
41. C. Gomez-Navarro, RT. Weitz, AM. Bittner, M. 
Scolari, A. Mews, M. Burghard, K. Kern. Electronic 
transport properties of indivisual chemically reduced 
graphene oxide sheets, Nano Letters, 2007, 7, 3499-
3503. 
42. A. Ganguly, S. Sharma, P. Papakonstantinou, J. 
Hamilton. Probing the thermal deoxygenation of 
graphene oxide using high-resolution in situ X-ray 
based spectroscopies, J. Phys. Chem. C, 2011, 11, 
17009-17019. 
43. A. Viswanathan, AN. Shetty. Effect of dopants on the 
energy storage performance of reduced graphene 
oxide/polyaniline nanocomposite, Electrochim. Acta, 
2019, 327, 135026. 
44. S. Sahoo, PK. Sahoo, A. Sharma, AK. Satpati. 
Interfacial polymerized rGO/MnFe2O4/polyaniline 
fibrous nanocomposite supported glassy carbon 
electrode for selective and ultrasensitive detection of 
nitrite, Sens. Actuators B Chem., 2020, 309, 127763. 
45. M. Velicky, DF. Bradley, AJ. Cooper, EW. Hill, IA. 
Kinloch, A. Mishchenko, KS. Novoselov, HV. 
Patten, PS. Toth, AT. Valota, SD. Worrall, RAW. 
Dryfe. Electron transfer kientics on mono- and 
multilayer graphene, ACS Nano, 2014, 8, 10089-
10100. 
46. M. Ge, H. Hao, Q. Lv, J. Wu, W. Li. Hierarchical 
nanocomposite that couples nitrogen-doped graphene 
with aligned PANi cores arrays for high-performance 
Vietnam Journal of Chemistry Vu Thi Thu et al. 
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 262 
supercapacitor, Electrochim. Acta, 2020, 330, 
135236. 
47. H. Cui, W. Wu, M. Li, X. Song, Y. Lv, T. Zhang. A 
highly stable acetylcholinesterase biosensor based on 
chitosan - TiO2 - graphene nanocomposites for 
detection of organophosphate pesticides, Biosens. 
Bioelectron., 2018, 99, 223-229. 
48. X. Lu, L. Tao, D. Song, Y. Li, F. Gao. Bimetallic 
Pd@Au nanorods based ultrasensitive 
acetylcholinesterase biosensor for determination of 
organophosphate pesticides, Sens. Actuators B 
Chem., 2018, 255, 2575-2581. 
49. HD. Cancar, S. Soylemez, Y. Akpinar, M. Kesik, S. 
Goker, G. Gunbas, M. Volkan, L. Toppare, A Novel 
Acetylcholinesterase Biosensor: Core-Shell Magnetic 
Nanoparticles Incorporating a Conjugated Polymer 
for the Detection of Organophosphorus Pesticides, 
ACS Appl. Mater. Interfaces, 2016, 8, 8058-8067. 
50. A. Ivanov, R. Davletshina, I. Sharafieva, G. Evtugyn. 
Electrochemical biosensor based on polyelectrolyte 
complexes for the determination of reversible 
inhibitors of acetylcholinesterase, Talanta, 2019, 194, 
723-730. 
Corresponding authors: Vu Thi Thu 
University of Science and Technology of Hanoi (USTH) 
Vietnam Academy of Science and Technology (VAST) 
18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 
E-mail: thuvu.edu86@gmail.com / vu-thi.thu@usth.edu.vn. 
Tran Dai Lam 
Institute of Tropical Technology (ITT) 
Vietnam Academy of Science and Technology (VAST) 
18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 
E-mail: trandailam@gmail.com.