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

Cite this paper: Vietnam J. Chem., 2021, 59(2), 253-262

DOI: 10.1002/vjch.202000158

Article

Acetylcholinesterase sensor based on PANi/rGO film electrochemically

grown on screen-printed electrodes

Ly Cong Thanh¹, Dau Thi Ngoc Nga², Nguyen Viet Bao Lam³, Pham Do Chung³, Le Thi Thanh Nhi⁴,

Le Hoang Sinh⁴, Vu Thi Thu^2*, Tran Dai Lam^5*

¹Hanoi University of Pharmacy (HUP), 15-17 Le Thanh Tong, Hoan Kiem, Hanoi 10000, Viet Nam

²University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology

(VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam

³Hanoi National University of Education (HNUE), 134-136 Xuan Thuy, Cau Giay, Hanoi 10000, Viet Nam

⁴Duy Tan University (DTU), 03 Quang Trung, Da Nang 50000, Viet Nam

⁵Institute 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

biomolecules (i.e. enzymes) is often utilized in

electrochemical biosensors. Interestingly, PANi has

Hybrid films which combined biocompatible three different chemical states that can be tuned

polymers and highly conductive inorganic electrochemically^[6,7]

and sensitive to

nanomaterials have recently gained many attentions protonation/deprotonation process.^[8]Also, the

in sensing and electronic applications. Among well- presence of amino groups in polymer chains of

known conducting nanomaterials, graphene and its PANi make it becomes one favorable transducing

derivatives with extraordinary conductivity, platform to immobilize enzymes. Probably, the

mechanical stability and flexibility are the best hybrid structures based on PANi and carbonaceous

candidates that meet many critical requirements of materials should have inherited the mentioned

electrochemical sensing systems.^[1]Especially, benefits of these two materials.

reduced graphene oxide (rGO) is the most frequently

Several research groups have demonstrated

used since it provides many behaviors similar with potential applications of hybrid films based on

graphene and can be easily produced at large carbonaceous nanomaterials with PANi. Depending

scale^[2,3]through solution-based approaches and on the purpose of the application, these hybrid films

combined with other materials in composites.^[4,5]were grown either in composite structure or bilayer

Meanwhile, polyaniline (PANi) with good architecture. In the beginning, composite films based

conductivity, high porosity, and good adhesion to on graphene derivatives and PANi were mainly

Vietnam Journal of Chemistry

Vu Thi Thu et al.

utilized

for

developing

high-performance requires long procedure and complex instrument. In

supercapacitors in flexible energy storage devices.^[9-this work, rGO/PANi will be prepared on low-cost

11]

These hybrid composites also show high anti- screen printed electrode (SPE) using a simple

corrosion behavior.^[12]Recently, the layer-by-layer electrochemical process. Some details on growth

structure of hybrid films made of conducting mechanism of the hybrid film will be revealed. The

polymers and carbonaceous materials has drawn as-prepared hybrid film will be later utilized as a

more attentions. The assembly of the two distinct transducing platform to load acetylcholinesterase

materials in two separated layers allows better (AChE) and ready for monitoring acetylthiocholine

control in their thickness and homogeneity. The use (ATCh) - one important neurotransmitter involved in

of graphitic material as one supporting layer nervous communication.

provides the solution to overcome insulating nature

and structural shrinkage of PANi in dedoping 2. MATERIALS AND METHODS

states.^[13,14]Moreover, the addition of soft PANi

material make carbonaceous materials become less

rigid and more biocompatible. For instance, the

2.1. Chemicals

PANi ad-layer electrodeposited on graphitic Graphite powder, aniline (C₆H₅NH₂), sulfuric acid

electrodes has been shown to improve voltammetric (H₂SO₄), potassium permanganate (KMnO₄) were

signals during analysis of redox probes.^[15]purchased

from

Sigma-Aldrich,

USA.

PANi/graphene bilayer with good conductivity and Acetylthiocholine (ATCh), acetylcholinesterase

fast electron transfer has been shown to be profitable (AChE), phosphate buffered saline (PBS),

in electrochemical immunosensors for tracing neuro- glutaraldehyde (GA) were also from Sigma-Aldrich,

toxins.^[16]PANi/rGO bilayer was utilized as one pH- USA. Screen printed carbon electrodes (SPE) (Φ = 3

sensitive membrane to sense protons released from mm) were from Quansense, Thailand.

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.

2.2. Apparatus

Electrochemical experiments were conducted on an

Many

neurodegenerative

diseases

(i.e, AUTOLAB PGSTAT302N workstation (Metrohm,

Alzheimer’s disease and Parkinson’s disease) are the Netherlands). FE-SEM images (Field Emission

associated with the degeneration of the cholinergic Scanning Electron Microscopy) were captured on a

system that is caused by abnormal AChE activity. S-4800 system (Hitachi, Japan). ATR-FTIR spectra

Therefore, it is essential to develop realiable tools (Attenuated total reflection Fourier Transform

for monitoring the activities of AChE enzyme as Infrared spectroscopy) of the films were studied on a

well as screening their inhibitors. Acetylcholin- Shimadzu spectrometer (IR-Tracer 100). The

esterase sensors based on optical approaches^[18-20]crystalline structure of powder samples was verified

offer facile preparation and visual detection which by Raman spectroscopy on a Horiba spectrometer

are compatible for in-situ analysis. But using 532 nm excitation. X-ray photoelectron

acetylcholinesterase(AChE) electrochemical sensors spectroscopy (XPS) spectra were recorded on a

are still more preferable^[21]due to their good Thermo ESCALAB spectrometer (USA) using

sensitivity and their ability to be integrated onto employing a monochromic AlK_αsource at 1486.6

electronic devices. Metallic nanoparticles with good eV.

electrical conductivity and intrinsic electrocatalytic

activity have been previously employed to ensure

high sensitivity of enzymatic electrochemical

2.3. Synthesis of graphene oxide

sensors.^[22,23]Recently, carbonaceous materials are The graphitic flakes (200 mg) were oxidized using

gaining more attentions due to their high strong oxidizing agents, namely, KMnO₄(1 g) and

conductivity and good bioacompatibility.^[24-27]

H₂SO₄(30 mL) at 60 ^oC. After 24 hours, the reaction

In our research group, we have developed AChE solution was cooled down to room temperature and

electrochemical sensor based on graphene flakes left for two more days. The cooled solution with

modified with iron oxide nanoparticles.^[28]In another dark color was centrifuged at 8000 rpm. Then, the

work, AChE sensor was manufactured from solid precipitate was thoroughly rinsed until a mild

carbonnanotubes modified with thiophene polymer pH was obtained. Finally, the gained product was

o

and gold nanoparticles.^[29]In both cases, the dried at 60 C in an oven. More details on synthesis

carbonaceous materials have been synthesized using of graphene oxide (GO) were given in our previous

chemical vapor deposition (CVD) process which report.^[30]

Vietnam Journal of Chemistry

Acetylcholinesterase sensor based on…

mode (defect) are also observed at 2691 and 2934

cm^-1. Furthermore, the ratio between intensities of

2.4. Electrodeposition of PANi/rGO films

Cyclic voltammetry method is an approach able to two main peaks I_D/I_Gwas determined to be 0.95.

deposit thin films with controllable thickness and This is a clear evidence to demontrate high oxidation

uniform morphology. Carbon screen-printed degree of graphitic material. The crystalline size of

electrodes (on plastic substrates) which are suitable

for flexible and disposable biosensors are chosen in

our experiments.

(

)

graphitic flakes (evaluated from

) was estimated to be 20.23 nm and

(

⁄ )

1 mg.mL^-1GO dispersion in PBS (pH 7.4, 0.1x)

was used as deposition solution to electrodeposit

rGO film. In general, the negatively charged GO

flakes with abundant oxygenated functional groups

(OFGs) can be easily exfoliated due to electrostatic

respulsion. However, the use of electrolyte

containing anions and cations which is mandatory

for electrodeposition process might cause π-π

stacking of these exfoliated flakes. For this reason,

the precursor solution was sonicated for at least 30

min before use in order to obtain a well-dispersed

suspension of GO. The GO was directly reduced and

deposited on bare SPE electrode by using cyclic

voltammetry method at potentials ranging from -0.2

to -1.0 V with number of cycles and scan rate were

set to be 10 and 50 mV.s^-1, respectively.

190.19 nm for GO (I_D/I_G= 0.95) and graphite

powder (I_D/I_G= 0.1), respectively. This reduction in

the average size of graphitic domains is probably

resulted from structural disorder of sp³hybridized

carbon atoms during harsh oxidation process in

presence of strong oxidizing agents.

3.2. Growth of PANi/rGO bilayer

3.2.1. Electrodeposition of rGO film onto SPE

The electroreduction and direct deposition of GO

onto SPE using cyclic voltammetry (CV) method in

aqueous condition is shown in figure 1. The

sweeping potentials were chosen in the range from

-0.2 V to -1.0 mV in order to avoid hydrogen

evolution and possible reoxidation of carbonaceous

materials at more positive potentials.^[32]PBS buffer

(0.1 X) with neutral pH and diluted ion

concentrations (13.7 mM NaCl, 0.27 mM KCl, 1

mM Na₂HPO₄, 0.18 mM KH₂PO₄) was used as

electrolyte to limit the destabilisation of suspended

GO flakes at too high concentrations of ions. Due to

the dispersability of GO in water are typically from

1 to 4 mg.mL^-1, the concentration of GO precusor

was chosen to be 1 mg.mL^-1. The formation of black

rGO thin film directly deposited on the working

electrode can be easily observed by naked eyes.

A typical CV curve for electrodeposition of rGO

was obtained with one irreversible broad reduction

peak at -900 mV (vs Ag/AgCl) which occurred in

the 1^stcycle but disappeared in next scans (figure 1).

It is well-known that this peak is relevant to the

reduction of the oxygenated moieties on GO flakes.

The crossover (around -780 mV) in the 1^stcycle

during the electrodeposition of GO is resulted from

intrinsically poor conductivity of carbon SPE.

PANi was electrodeposited by sweeping as-

prepared rGO/SPE electrode in 0.03 M aniline

solution prepared in acidic 0.5 M H₂SO₄at

potentials from -200 mV to +900 mV. The number

of cycles and scan rate were set to be 5 and 50

mV.s^-1, respectively.

2.5. Sensing performances of acetylcholinesterase

sensor based on PANi/rGO

AChE enzyme (20 IU) was immobilized onto

sensing platform using glutaraldehyde vapor (GA)

as cross-linking agent at 4⁰C for 90 min.

Amperometric responses of the acetylcholinesterase

sensors based on PANi/rGO/SPE were recorded

upon successive injection of acetylthiocholine

solution (5 mM, 2 µL) to a static PBS drop (50 µL)

covered totally the three electrodes of SPE. The

applied voltage was set to be +300 mV (vs

Ag/AgCl).

3. RESULTS AND DISCUSSION

According to the widely accepted structure

model proposed by Lerf-Klinowski (figure S2),

major oxygenated functional groups (OFGs) in GO

materials mainly include hydroxyl and epoxy groups

at basal planes, carbonyl groups at flake edges that

can contribute to several irreversible electrochemical

processes.^[33,34]It was also reported that the

reduction of carbonyl groups at the graphitic edges

occurs at more negative potentials (-1050 to -1220

mV) whereas that of basal epoxy moieties occurs at

3.1. Structural behaviors of graphene oxide

The crystalline structure of GO material was

examined using Raman technique (figure S1). The

curves displayed two prestigious peaks at 1348 and

1593 cm^-1relevant to D mode (A_1g) and G mode

(E_2g), respectively.^[31]The two peaks relevant to 2D

mode (double resonance transitions) and (D+G)

Vietnam Journal of Chemistry

Vu Thi Thu et al.

more positive potentials (-876 to -1120 mV).^[30]As surface. Second, the functionalization of basal

seen from cyclic voltammograms (figure 1), there is planes with these negatively charged molecules will

only one well-defined reduction peak which is probably facilitate the intercalation of water

probably assigned to the reduction of epoxy groups molecules and soluble molecules into these gaps,^[33]

at basal planes. This reduction process (figure S3) thus accelerate once more the reduction and

will probably restore more sp²hybridized carbon deposition process of carbonaceous flakes. On the

atoms and might also generate more hydroxyl other hand, the grown rGO film should be very

functional groups, thus much improve electrical compact and durable. Finally, the hydrophilization

conductivity as well as hydrophilicity of the of basal planes with these hydroxyl moieties will

electrode surface at the same time.^[34]

probably provide nucleation sites that can easily

adsorb aniline monomer and then facilitate the

growth of polymeric ad-layer on top of GO

film.^[12,36]

3.2.2. Electrodepostion of PANi film onto rGO/SPE

The CV curves recorded during polymerization of

aniline on rGO/SPE electrode using cyclic

voltammetric method is shown in Figure 2. Since the

protonation is essential in polymerization of

aniline,^[36]the electrodepostion of PANi is conducted

in a diluted acidic solution. The process was stopped

after 5 cycles at 0 V to ensure the high conductivity

of synthesized film by achieving a moderately thin

PANi layer in emeraldine form.^[37]

A typical CV curve for electropolymerization of

PANi was obtained with two anodic waves located

at +266 mV and +752 mV relevant to transition

from leucomeraldine to emeraldine salt and

formation of fully doped perningraniline,

respectively.^[6]Similar to any electrodeposition

process of conducting polymers, the intensities of

those two peaks increased consecutively with

number of scans. It is worth to notice that the

inversion current (current at switched potential of

+900 mV) was found to be decreased, indicating a

progressive nucleation which will lead to a porous

structure of polymer film.^[6]It was generally

accepted that the growth of electrodeposited PANi

film is a nucleation process.^[36]In aqueous medium

Figure 1: Cyclic voltammograms recorded during

electrodeposition of GO onto SPE

2-

containing small doping counter ions (i.e. SO₄), the

electrodeposition is initiated by three-dimensional

progressive nucleation and followed by prolongation

of one-dimensional polymer branches. Herein, the

polymer chains must have been nucleated

progressively on rGO modified electrodes and then

grown in branch-like structure.

The growth of PANi film onto rGO modified

electrodes should be more favorable compared to

bare electrodes. First of all, the carbonaceous

substrate provided additional surfaces for the

adsorption of aniline monomers and oligomers.^[13]

As mentioned above (section 3.2.1), the existence of

previously deposited rGO layer with high

compactness might offer more nucleation sites, thus

Figure 2: Cyclic voltammograms recorded during

electrodeposition of PANi onto rGO/SPE

High concentration of hydroxyl groups at basal

planes of graphitic flakes provides many benefits.

First, the reduction of epoxy molecules to hydroxyl

molecules was accompanied with direct deposition

of graphitic material onto the electrode surface. GO

flakes accumulated on the electrode surface can be

reduced and spontaneously solidified, whereas GO

flakes partially reduced in electrolyte keep migrating

upon the driving of electric field to electrode

Vietnam Journal of Chemistry

Acetylcholinesterase sensor based on…

increased the disposition rate of PANi.^[36]Last but casted GO film was also prepared and characterized.

not least, the adhesion of PANi with amino groups The C:O ratio (see table 1) was determined to be

onto basal planes of graphitic flakes with abundant 1.70, 2.044, and 2.313, and 2.025 for GO, rGO,

OFGs should be strengthened by cross-linking PANi and PANi/rGO films, respectively. On the

bonds^[14,17]as well as π-π stacking interaction other hand, the oxygen content was decreased after

between these two materials.

electrochemical reduction of GO, but slightly

increased in presence of PANi top layer. It is

obvious that the atomic percentage of oxygen atoms

must be decreased after reducing OFGs at basal

3.3. Morphological and structural behaviors of

PANi/rGO/SPE

planes of graphitic flakes. The existance of doping

2-

Figure 3 illustrates the surface morphologies of rGO counter ions SO₄on polymer chains in top layer^[40]

and PANi/rGO films examined by FE-SEM. It is and the unhealed lattice defects in underlying

obvious that the rGO film with multi-layered graphitic flakes^[41]are responsible for slight increase

structure of stacked flakes shows a smooth surface in atomic percentage of oxygen atoms in PANi/rGO

with several wrinkles. Meanwhile, PANi/rGO film (compared to individual rGO and PANi films).

shows a micropourous network which is valuable for

electron transport processes. The polymer chains are

formed in fibrillar-like structure which is intrinsic

architecture of PANi film electrodeposited in

aqueous conditions. This result is consistent with the

progressive nucleation mechanism of PANi film as

mentioned in section 3.2.2. Such a highly porous 3D

architecture of PANi/rGO bilayer is very promising

transducing

platform

in

enzyme

based

electrochemical sensors for its accelerated electron

transfer rate and improved adhesion to biomolecules.

Figure 4: ATR-FTIR spectra of rGO (red) and

PANi/rGO (black) films

Table 1: Analysis results derived from XPS spectra

Sample

C/O

GO

rGO

PANi

1.699

2.044

2.313

2.025

PANi/rGO

Figure 3: FE-SEM images of bare SPE (A),

rGO/SPE (B) and PANi/rGO/SPE films (C)

C 1s core-shell spectrum of GO drop-casted film

shows strong signals ascribed to graphitic carbon

atoms (284.7 eV) and oxidized carbon species (C-O

IR spectra of rGO and PANi/rGO films are 286.9 eV, C=O 288.5 eV).^[14]Upon electrochemical

given in figure 4. The stretching vibrations of treatment, the peak associated with graphitic carbon

hybridized and oxygenated carbon atoms in rGO atoms becomes prominent while the peaks

films were found at 1600 and 990 cm^-1. Meanwhile, ascertained to OFGs becomes weaker (see table 1).

the characteristic vibrations of non-nitrogenated The most significant change in XPS spectra is

(1572, 1489, 821 cm^-1) and nitrogenated (1298 and observed in the concentration of C-O groups (epoxy)

1113 cm^-1) moieties of polyaniline in emeraldine which is in good agreement with CV records. The C-

form were also clearly observed.^[37-39]

O/C-C ratio was determined to be 0.925 for GO film

XPS spectra of rGO and PANi/rGO were but only 0.736 for rGO film (decreased by 20 %). In

investigated (figure S4). For comparison, drop- the same time, O1s spectrum of rGO film reveals

Vietnam Journal of Chemistry

Vu Thi Thu et al.

one peak located at high energy level (534.5 eV)

3.4. Electrochemical behaviors of PANi/rGO/SPE

which is in visual in O 1s spectra of other films. The

appearance of this peak is probably ascribed to The charge transfer kinetics at modified electrodes

phenolic groups at basal planes and/or intercalated was examined using cyclic voltammetry in 1mM

3-/4-

water molecules.^[42]These results have provided Fe(CN)₆

solution (figure 5). For bare electrode,

clear evidences to demonstrate that the the two redox peaks occurred with peak separation

electrochemical reduction of GO precursor has of 440 mV which is much larger than usual glassy

mainly happened at basal epoxy groups.

carbon electrodes due to the poor conductivity of

N 1s signals for PANi/rGO film can be carbon SPE. This result is in agreement with the

deconvoluted to assign benzenoid amine –NH- observation of a crossover current in the first cycle

(399.3 eV) and cationic radical N⁺(401.3 eV).^[14,43,44]recorded during electrodeposition of rGO onto SPE

The first peak relevant to amine group is located at (section 3.2.1). Modification of SPE electrode with

higher binding energy compared to neutral amine (at rGO material has not only increased the peak

399.5 eV),^[37]indicating the occurence of partially intensities by 1.3 times but also much shortened

charged nitrogen in electrodeposited polymer chains. peak separation by 240 mV (table 2). This is

The second peak is typical for PANi at doped obviously resulted from good conductivity^[41]and

state.^[40]These results have confirmed the presence fast electron transfer rate^[45]at the basal planes of

of PANi layer on top of rGO film. The N⁺/N ratio in rGO. Also, it was generally accepted that the

PANi/rGO film was found to be 0.492 which is presence of carbonaceous nanomaterials might lower

slightly lower than that obtained on PANi film energy required for electrochemical reactions

(0.583). This indicates that the PANi chains might occurring at electrode surface. Even the rate of

have been dedoped partially in presence of charge transport at rGO film is lower by several

negatively charged moieties from rGO layer. orders compared to pristine graphene material,^[41]it

Nevertheless, the proton doping level of PANi is still a promising candidate for electronic devices.

chains in PANi/rGO bilayer is still relatively high, Moreover, rGO film can further be used as a

and thus affords a good electrical conductivity.^[40]

supporting layer to accelerate the growth of

appropriate organic ad-layer. When PANi is

deposited on top of rGO, the peak intensities were

improved (3.8 times higher than SPE, 2.9 times

higher than rGO/SPE) and the peak separation was

continued to be decreased to 135 mV). It was

reported that the combination of highly conductive

carbonaceous materials and highly porous PANi can

enhance ion diffusion and charge transport which is

very profitable for further applications in

electrochemical

sensors

as

well

as

supercapacitors.^[46]

2100

1800

1500

1200

900

600

300

0

Figure 5: Electrochemical behaviors of bare SPE

(black) and SPE modified with rGO (red),

rGO/PANi (blue)

Table 2: Electrochemical behaviors of rGO and

PANi/rGO films

Peak separation

Intensity

(µA)

10

Sample

(mV)

440

400 600 800 1000 1200 1400 1600 1800 2000

SPE

t (s)

rGO/SPE

PANi/rGO/SPE

200

135

13

38

Figure 6: Current responses recorded on as-prepared

acetylcholinesterase sensor

Vietnam Journal of Chemistry

Acetylcholinesterase sensor based on…

polymerization of nanofibrillar-like polyaniline

films on top of rGO film. The acetylcholinesterase

3.5. Performances of Acetylcholinesterase sensor

The sensing performance of acetylcholinesterase sensor based on PANi/rGO bilayer was built as a

sensor was examined using chrono amperometric proof-of-concept to demonstrate its potential

(CA) method with the applied voltage of +300 mV biosensing application. In our future work, more

(figure 6). Upon the injection of an aliquot of ATCh, details on the effects of reduction degree on

the current response increased rapidly with response concentration of OFGs (i.e. hydroxyl groups) on

time less than 10s. The response current of the morphology and charge transfer kinetics of hybrid

sensor was increased with the increasing films based on rGO and several conducting

concentration of ATCh according to regression polymers will be studied.

equations: I (nA) = 1845.3 C_ATCh(mM) - 289.5

(0.192 to 1.094 mM) (figure 7).

Table 3: Comparisons between AChE

electrochemical sensors

1800

1500

1200

900

600

300

0

Dection

limit

(µM)

K_m

(mM)

Linear

range

Configuration

Pd@Au/AChE

Ref.

4-124

µM

-

0.19 [20]

3.1 [37]

GCE/rGO/CS@ 0.1-9.0

TiO₂-CS/AChE

GCE/Pd@AuN

Rs/AChE-

CS/Nafion

Graphite

electrode/poly(F

BThF)/MNPs/A

ChE

mM

2-272

µM

-

0.207 [38]

0.125-

2.6

mM

0.2

0.4

0.6

0.8

1.0

1.2

6.66

0.731 [39]

2.16 [40]

C (mM)

Figure 7: Calibration of as-prepared

acetylcholinesterase sensor

GCE/PDDA/PS 1 µM-

-

S/AChE

10 mM

12.5-

112.5

µM

0.192-

1.094

mM

app

The apparent Michaelis - Menten constant K_m

GCE/Gr-

MNPs/AChE

was 0.728 mM (from Lineweaver-Burk relation).

The limit of detection (LOD) was determined from

standard deviation (after three measurements) and

slope of calibration curve to be 17.5 µM. These

obtained values are comparable to those previously

reported in other works (see table 3),^[47-50]showing

8.35

-

[41]

SPE/rGO/PANi/

AChE

This

work

17.5

0.728

good affinity of immobilized enzyme AChE for the 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.

targeted substrate ATCh. The developed PANi/rGO

bilayer is a universal electrochemical platform that

can be further applied to load many other biological

elements and ready to monitor different biological

processes, especially the ones that are pH sensitive.

Declaration of interest. The authors have no

financial interests to declare.

4. CONCLUSIONS

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.

PANi/rGO film with layer-by-layer structure was

successfully electro-deposited onto screen-printed

electrode with significantly improved electrical

conductivity and electron transfer kinetics. The

reduction of epoxy groups at basal planes was found

to be dominant during direct electrodeposition of

rGO film in aqueous medium at neutral pH.

Consequently, these hydrophilic flakes facilitate

progressive nucleation and accelerate the

Vietnam Journal of Chemistry

REFERENCES

Vu Thi Thu et al.

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

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.

ternary composite

graphene oxide@PANi

of

nanodiamonds/reduced

for electrochemical

capacitors, Chem. Phys., 2019, 526, 110461.

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.

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.

3. H. Wang, JT. Robinson, X. Li, H. Dai. Solvothermal

reduction of chemically exfoliated graphene sheets, J.

Am. Chem. Soc., 2009, 131, 9910-9911.

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.

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.

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.

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.

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.

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.

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.

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.

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

9. D. Liu, H. Wang, P. Du, W. Wei, Q. Wang, P. Liu.

Flexible and robust reduced graphene oxide/carbon

nanoparticles/polyaniline

composite films: Excellent candidates as free-

standing electrodes for high-performance

(RGO/CNs/PANI)

based

ratio

fluorescent

strategy

for

acetylcholinesterase activity sensing, Sens. Actuators

B Chem., 2020, 313, 128023.

supercapacitors, Electrochim. Acta, 2018, 259, 161-

169.

21. S. Kurbanoglu, C. Erkmen, B. Uslu. Frontiers in

electrochemical enzyme based biosensors for food

and drug analysis, Trends Anal. Chem., 2020, 124,

115809.

10. A. Aydinli, R. Yuksel, HE. Unalan. Vertically

aligned carbon nanotube - Polyaniline nanocomposite

supercapacitor electrodes, Int. J. Hydrog., 2018, 43,

18617-18625.

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.

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,

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…

organophosphorus pesticides detection, Sens. 35. A. Buchsteiner, A. Lerf, J. Pieper. Water dynamics in

Actuators B Chem., 2019, 284, 103-109.

graphite oxide investigated with neutron scattering, J.

Phys. Chem. B, 2006, 110, 22328-22338.

24. QT Hua, N. Ruecha, Y. Hiruta, D. Citterio.

Disposable electrochemical biosensor based on 36. I. Turyan, D. Mandler. Two-dimensional polyaniline

surface-modified screen-printed electrodes for

organophosphorus pesticide analysis, Anal. Methods,

2019, 11, 3439-3445.

thin film electrodeposited on a self-assembled

monolayer, J. Am. Chem. Soc., 1998, 120, 10733-

10742.

25. B. Zou, Y. Chu, J. Xia. Monocrotophos detection 37. Z. Mandic, L. Duic, F. Kovacicek. The influence of

with a bienzyme biosensor based on ionic liquid

modified carbon nanotubes, Anal. Bioanal. Chem.,

2019, 411, 2905-2914.

counter-ions on nucleation and growth of

electrochemically synthesized polyaniline film,

Electrochim. Acta, 1997, 42, 1389-1402.

26. J. Bao, T. Huang, Z. Wang, H. Yang, X. Geng, G. 38. X. Zhao, Y. You, S. Huang, F. Cheng, P. Chen, H.

Xu, M. Samalo, M. Sakinati, D. Huo, C. Hou. 3D Li, Y. Zhang. Facile construction of reduced

graphene/copper oxide nano-flowers based graphene oxide supported three-dimensional

acetylcholinesterase biosensor for sensitive detection

of organophosphate pesticides, Sens. Actuators B

Chem., 2019, 284, 95-101.

polyaniline/WO_2.72nanobelt-flower as a full solar

spectrum light response catalyst for efficient

photocatalytic conversion of bromate, Chemosphere,

2019, 222, 781-788.

27. S. Nagabooshanam, AT. John, S. Wadhwa, A.

Mathur, S. Krishnamurthy, LM. Bharadwaj. Electro- 39. R. Arukula, M. Vinothkannan, AR. Kim, DJ. Yoo.

deposited nano-webbed structures based on

polyaniline/multiwalled carbon nanotubes for

enzymatic detection of organophosphates, Food

Chem., 2020, 323, 126784.

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.

28. Dau TNN., Vu VH., Cao TT., Nguyen VC., Ly CT.,

Tran DL., Truong Thuan Nguyen Pham, Nguyen TL., 40. S. Gao, L. Zhang, Y. Qiao, P. Dong, J. Shi, S. Cao.

Benoit Piro, Vu TT. In-situ electrochemically

deposited Fe₃O₄nanoparticles onto graphene

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.

nanosheets

as

amperometric

amplifier

for

electrochemical biosensing applications, Sens.

Actuators B Chem., 2019, 283, 52-60.

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.

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.

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.

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.

43. A. Viswanathan, AN. Shetty. Effect of dopants on the

energy storage performance of reduced graphene

oxide/polyaniline nanocomposite, Electrochim. Acta,

2019, 327, 135026.

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.

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.

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.

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.

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 46. M. Ge, H. Hao, Q. Lv, J. Wu, W. Li. Hierarchical

graphene oxide, Electrochim. Acta, 2019, 304, 231-

238.

nanocomposite that couples nitrogen-doped graphene

with aligned PANi cores arrays for high-performance

Vietnam Journal of Chemistry

Vu Thi Thu et al.

supercapacitor, Electrochim. Acta, 2020, 330, 49. HD. Cancar, S. Soylemez, Y. Akpinar, M. Kesik, S.

135236.

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.

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.

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.

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

Chem., 2018, 255, 2575-2581.

B

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.