Analytical detection of paraoxon using acetylcholinesterase as an enzyme on polyaniline/ferric chloride composite film by potentiostatic method

Vikas.B. Deshmukha, Kiran.S. Paithankara,Urrmila.N. Shelkea, Vikas.K. Gadeb*

aDepartment of Physics Hutatma Rajguru Mahavidyalaya,Rajgurunagar, (M.S.) IndiaaDepartment of Physics Research Center,Ahmednagar College Ahmednagar (M.S.) India

bDepartment of Physics, Shri Anand College, Pathardi (M.S.) India

vbdeshmukhsir@gmail.com vikas_gade@yahoo.comAbstracts:

 In this paper, a novel responsive electrochemically synthesized composite film was used for

development of biosensor to determination of paraoxon. The sensor was fabricated by coating a

polyanilne (PANI) and ferric chloride (FeCl3) on indium tin oxide (ITO) by electrochemical

deposition process. The PANI/FeCl3 composite film plays the important roles to immobilized

acetylcholinestrase (AChE) to improve the electrochemical response. Under optimized condition,

the detection of paraoxon in a linear range from 0.1µM to 0.9µM was obtained and the detection

limits was 0.1µM. The resulting sensor was sensitive and highly stable for paraoxon. It shows

very good storage stability and recovery rate was 50% of its primary activity within 36 days

while keep at 6 0C.              

 Keywords: Polyaniline, Electrochemical, Sensor, Acetylcholinesterase, Paraoxon.

1. Introduction

Paraoxon, synthetic phosphorus plays an considerable task in new agricultural production for the

reason that it can protect from insects and agricultural pests or unwanted plants and to improve

plant quality [1-2] However paraoxon residue in fruits, food and environment has harmful to

human health due to its high toxicity, so novel responsive electrochemically synthesized sensor

was developed for the determination of paraoxon. Now a day, many methods have been used for

detection of pesticide such as ‘gas chromatography’ [3] ‘liquid chromatography’ [4] and ‘thin

layer chromatography’ [5] these methods are responsive and reliable but they usually suffer from

costly instrumentation and time consuming [6] compared with biosensor made by

electrochemical synthesis, it is sensitive, quick response and has low-cost, This sensor has wide

application in biological, agricultural and environmental analysis. However there are some

reports on the electrochemical detection of paraoxon such as Fabiana Arduini et.al [7] prepared

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‘an bounded screen-printed electrode customized with butyrylcholinesterase and Prussian blue

nanoparticles for detection of paraoxon whose linear range was 7×10−9M-4×10−8 M and detection

limit was 4× 10−9 M.’ Yan Ping et al employed ‘new AChE biosensor for detection of paraoxon

based on porous graphene oxide modified glass carbon electrode, the linear range was 10-45

ng/ml and detection limit was 1.58 ng/ml’ [8]. The electro-analytical method is sensitive but

shows poor selectivity therefore to develop electrochemical biosensor with high selectivity for

detection of paraoxon. Conducting polymer composite film means biosensor is a good choice for

detection of pesticides, it is independent device that integrates immobilized biological element

like enzyme [9-13].The enzyme based sensor is a good choice because of their simple synthesis

and measurement process, high responsive, short response time, sensitive, and reproducible. The

inhibition activity of enzyme is controlled by oxidation current by applied certain potential. To

study of the structural, electrical and optical properties of polymer films for immobilization point

of views AChE enzyme is easy immobilization on thin film for determination of pesticides [14].

AChE enzyme is excellent inhibitor for polymer film to produce sensor which is sensitive

microenvironment for bio-molecules and good bonding to the anlyte [15-16].  In present work

the electrochemical polymerization takes place due to redox reaction with suitable concentration

of aniline, H2SO4 and FeCl3 and Indium tin oxide (ITO), platinum foil and silver/silver chloride

(Ag/AgCl) electrodes are used in cell.  PANI/FeCl3 composite film is synthesized for

development of pesticide sensor by immobilization of AChE enzyme for clean with phosphate

buffer solution [17-18]. 

2. Experimental methods

2.1 Reagents

Aniline (98%), Paraoxon (99%), and Acetylcholinesterase was purchased from Sigma Aldrich,

Required chemicals and ITO Sheet purchased from Lobachemie Pvt. Ltd. Mumbai, India.

2.2. Apparatus

UV-visible spectroscopy-Analytic Jena specord 210 plus Wavelength (200 nm-800 nm), Fourier

transformation infrared radioscopy FTIR-6100 type, Scanning electron microscopy-FEI (Nova

NanoSEM 450) with Energy Dispersive Spectrometer (EDS), Single Crystal- X-Ray

Diffractometer (SC-XRD) was taken by Savitribai Phule Pune University, CIF (Central

Instrumentation Facility) Pune, India.

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2.3. Electrochemical Synthesis of PANI/FeCl3                

 Electrolytic solution was made by 0.5M H2SO4, 0.5M FeCl3 and 0.1N an aqueous solution of

aniline (98%) monomer in double distilled water in electrochemical cell [19-20]. The pH of

aqueous medium was maintained by using phosphate buffer solution. The electrochemical

polymerization of aniline monomer and FeCl3 was synthesized by galvanostatic method [21-24].

PANI film is composed with FeCl3 on ITO substrate. 

2.4. Synthesis of enzyme based biosensor AChE/PANI/FeCl3/ITO 

Pure 8.0 µL enzyme (AChE) was dropped on the surface of the PANI/FeCl3/ITO thin film (PBS)

and incubates it at 200C for 24 h. then rinse with distilled water, dried out and keep at 60C prior

to use. Then this electrode was prepared for electrochemical measurement, it constitute an actual

role from the composite surface for the effectiveness of the biosensor without the cross-linking

agents to build bonding to the active site of enzymes [25-26].  

 2.5. Electrochemical Measurement

The current response of the sensor for detection of pesticide was measured with a two-step

process. The AChE/PANI/FeCl3/ITO sensor was immersed in a cell contain 10 ml buffer

solution (pH 7.0) and observed current response was I0 then the sensor was incubated for 20 min.

in 0.5M of standard solution of paraoxon. After the incubation the sensor was washed with

buffer solution, then immersed to the cell containing buffer solution pH 7.0 and measured current

response which was I1. The inhibition rate of the pesticides was calculated by following formula.

∆I (%) = (I0-I1)/I0 X100

Where, ∆I= inhabited rate, I0= initial current response of sensor without paraoxon and I1 = current

response in standard solution of paraoxon [27].

2.6. Acetylcholinestrase reactivation

For the purpose of second time use and economy, PANI/FeCl3/ITO electrode was recovered by

pyridine 2-aldoxime methochloride (2-PAM), the sensor was free to pesticides, it was wash with

PBS (pH-7) and distilled water and reactivated with 4.0 mM 2-PAM for 10 minutes, then

transferred to the electrochemical cell of 10 ml PBS (pH-7) under slow magnetic stirring.

Paraoxon was added and the peak current was recorded as I2. The reactivation efficiency (R %) is

calculated as the following equation [28].                                 

R % = (I2/I0) ×100%

Where, I2= the current response after AChE reactivation.

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3. Result and Discussion  

3.1. Ultraviolet–Visible spectroscopy 

The absorption spectrum of PANI/FeCl3/ITO film was recorded at room temperature by

Analytic Jena specord210 plus spectrophotometer with wavelength range 200 -800 nm as shown

in fig.1 the spectra shows three peaks at around 264nm, 331nm and 491nm. The peak at around

264 nm shows π→ π* transitions which is available in compounds with unsaturated centers and

aromatics compounds. Second peak 331nm absorption is responsible for bi-polaron state and

peak 491 nm shows charge transfer bands indicates conductivity of thin film having band gap

energy is 2.56 eV. The peak 491 nm indicates aniline is more doped has resulted in stronger

absorption. The absorption spectrum observed for synthesized composite film has good

agreement with the earlier reported work [29]. This shows excellent semblance with the

polymerization potential.

3.2. Fourier Transformation Infrared Radiation (FTIR) analysis 

The FTIR spectrum of PANI/FeCl3/ITO composite film with immobilization of AChE enzyme is

shown in fig. 2 the spectra show various peaks, the peak at 3325.33 cm -1 tends to N-H stretching.

The peaks at 1230.38 and 1338.19 cm-1 which is assigned to evidence of the presence of anion in

the polymer film i.e. S=O stretching in sulfonate. The peak having wave number 1709.33 cm-1

shows vibration bands (C=O) these peaks indicate to the characteristic for aniline it shows really

nice agreement with previous reported work [30]. Thus the FTIR spectral results confirm the

existence of polyaniline.

3.3. X-ray diffraction 

X-ray spectroscopy of PANI/FeCl3/ITO film is shown in fig.3 the intensity of observed peaks is

indicates that the composites film is developed using di and tri basic acid solutions compare with

the monobasic acid. The report of the characteristic peak of PANI/FeCl3/ITO at 2Ɵ =25.110 the

obtained pattern shows a mostly amorphous material with few crystalline phases.

3.4. Scanning Electron Microscopy and Energy dispersive X-ray spectroscopy 

The SEM images of PANI/FeCl3/ITO of synthesized thin films with and without immobilization

of enzyme are shown in fig.4 (a, b, c and d) having magnifications 30KX and 50KX. Image a, b

shows porous morphology of composite film. Such nature is easily supportive for immobilization

of enzymes for the biosensor application. Image c and d show morphology changed with enzyme

immobilized onto the modified electrode further, uniform and granular coating of the enzyme

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proved that the sensor electrode before immobilization serves as a very good host-guest platform

for bimolecular immobilization. Fig. 4.e and f shows E-DAX spectrum and graphical

representation of electrode. It indicates composition of various chemical compounds used for

synthesized film. E-DAX image also finds parentages of chemical elements present in a sample.

4. Optimization parameters of experimental 

4.1. Effect of potential

The potential of the PBS solution was measured by AChE/PANI/FeCl3/ITO electrode with

current. Fig.5 shows the current increases rapidly with increase in potential which indicates that

the response of the AChE electrode was controlled by the electrochemical method. The rate of

redox reaction is related to the concentration of electro active group, the pH value of solution and

applied potential [31]. The response of the electrode at higher potential is decreased but at 0.9V,

the current was nearly steady at which the rate of limiting process of enzyme, diffusion-control

of H2O2 and substrate [32]. So we had set the potential at 0.9V for the further studies of sensor

electrode.

4.2. Effect of pH 

The pH of the electrolytic solution was adjusted by using HCl and NaOH. The pH reading of

solution is measured by taking the range from 1 to 9. It prevents loss of the enzyme activity

under polymerization conditions [33].Therefore pH is important for enzymatic sensor response

of the sampling solution. The effect of pH on the performance of the AChE electrode was

studied with 0.1 M PBS with 0.5 µM paraoxon. The current is measured at 0.9V by various pH

values as is shown in fig.6. In electrochemical polymerization the doping response for

electrolytic substance and dopants was quite better at pH range from 2 to 5 and the peak current

occur at pH 7 [34].

4.3. Current response 

The current response of AChE/PANI/FeCl3/ITO sensor was measured for various paraoxon

concentrations with time set at 0.9V constant potential. The current is measured in the linear

range 0.1 µM to 0.9 µM of paraoxon concentration. It was found that, the current increases with

increasing paraoxon concentration as shown in fig.7 In the present case, the enzyme is uniformly

immobilized on the composite film so the reaction takes place mainly on the surface of the film

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in the lower concentration and the dispersion taking place at the same time at higher

concentrations delays the response time. 

4.4. Michaelis–Menten constant (Km)

Michaelis–Menten constant shows the concentration of the substrate when reaction velocity is

equal to one half of the maximum velocity. The relationship between current-1 against paraoxon-1

concentration in 0.1 M PBS is shown in Fig.8 at the maximum current (Imax) was 29 μA. The

value of Km is 0.9 it depends on immobilization of enzyme, low value of Km shows reaction

approaches more rapidly and faster response of the sensor.

4.5. Effect of incubation time

The enzyme activity of pesticides sensor was tested for incubation times (0–20 min) in a 0.5µM

pesticide solution. It was observed that enzyme inhibition increased with incubation period until

getting a stable level as shown in fig.9 however the decrease in activity was less prominent after

10 minutes for pesticide so incubation time was 10 min. which is better than earlier works [35].

4.6. Reactivation of the AChE electrode with 2-pyridine aldoximemethiodide (2-PAM)

The sensor was reactivated by cleaned with PBS and immersed in a 4.0 mML -1solution of 2-

PAM, 90% activity of AChE was restored of its original value by 2-PAM treatment for 15 min.

i.e. irreversible inhibition of AChE pesticides electrode was reactivated by this process.

Table1. Comparison of performance of various sensor electrodes for pesticide detection

     

Sensor electrode Pesticide (Analyte)

Linear range  Detection limit

Inhibition time (min)

References 

Au/PANI modified CPE Atrazine 1-18µMol/L 0.3µMol/L 

- 24

AChE/Fe3O4/Gr/ SPE Chlorpyrifos 0.05-100  μg/L 0.02 μg/L 15 26

AuNps/MPS/Au/AChE Carbamate 0.003-3.0µM 1.0nM 10 32

AChE/SnO2cMWCNTs/Cu MethylParathion

1-160 𝜇M 0.1𝜇M - 31

PANI/AuNPs/Pt/AChE Paraoxon 3.6 × 10−7-3.6 × 10−4 𝜇M

0.026 × 10−5 𝜇M20 34

BChE immobilized on PBNPs/SPE

Paraoxon  7 × 10−9M-4 × 10−8 M

4 × 10−9 M 10 7

6

AChE/HGO/GCE Paraoxon 10-45 ng/ml 1.58 ng/ml 10 8

AChE/PANI/FeCl3/ITO Paraoxon 0.1µM- 0.9µM 0.1µM 10 This work

5. Storage stability

The sensor shows long term stability for the suitable use of its application. The storage stability

of the sensor was tested for 6 weeks of storage in 0.1 M PBS (pH 7.0) at 25°C as shown in

fig.10. There was decrease in sensitivity of the sensor of about 10% from the primary value for

every week, It is observed that a very good conservation of the bioactivity than other work.

6. Conclusion

The stability of electrode was enhanced by using ferric chloride as to immobilize the AChE. The

AChE/PANI/FeCl3/ITO electrode shows excellent conductivity, simple and uniform deposition

on surface area of film and good stability as well as sensor is highly responsive, wide detection

range and response time is short. This is inexpensive and simple fabrication method of

AChE/PANI/FeCl3/ITO sensor. The method is used to immobilize enzymes to build a range of

sensors as well as to develop other biological molecules, such as DNA and antibody of sensor.

Acknowledgements

Central Instrumentation Facilities (CIF) Savitribai Phule Pune University (SPPU), Department of

Chemistry and Physics SPPU, Pune, (India) was provided characterization facility. Physics

research centre, Ahmednagar College, Ahmednagar (M.S.) India was provided laboratory

facility.

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Figures title

Fig .1 UV–Vis studies of PANI/FeCl3/ITO Electrode

Fig. 2 FTIR studies PANI/FeCl3/ITO based Electrode.

Fig. 3 XRD of PANI/FeCl3/ITO

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Fig. 4.a SEM of PANI/FeCl3/ITO film without enzyme   (Mag. 30 KX)

Fig. 4.b SEM of PANI/FeCl3/ITO film without enzyme (Mag. 50 KX)  

Fig.4.c SEM of PANI/FeCl3/ITO film with enzyme (Mag. 30 KX)   

Fig.4.d SEM of PANI/FeCl3/ITO film with enzyme (Mag. 50 KX)

Fig.4.e E-DAX of PANI/FeCl3/ITO film

Fig. 5 Current verses potential curves for the AChE/PANI/FeCl3/ITO sensor in 0.1M PBS

Fig. 6 Effect of pH on the AChE/PANI/FeCl3/ITO sensor response of paraoxon

Fig. 7 Current response curve of AChE/PANI/FeCl3/ITO sensor

Fig.8 Michaelis–Menten constant (Km) for the AChE/PANI/FeCl3/ITO sensor in 0.1M PBS

Fig.9 Effect of inhibition time on paraoxon (in 0.5 µML−1) at 0.9 V vs. Reference electrode

Fig.10 Storage stability of the AChE/PANI/FeCl3/ITO sensor in 0.1M PBS

Fig .1 UV–Vis studies of PANI/FeCl3/ITO Electrode

     Fig. 2 FTIR studies PANI/FeCl3/ITO based Electrode

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Fig. 3 XRD of PANI/FeCl3/ITO (Intensity-1237 and 2θ-25.61)

     Fig.4 (a) SEM of PANI/FeCl3/ITO film without enzyme   (Mag. 30 KX), (b) SEM of

PANI/FeCl3/ITO film without enzyme (Mag. 50 KX)     

      

Fig.4 (c) SEM of PANI/FeCl3/ITO film with enzyme   (Mag. 30 KX), (d) SEM of

PANI/FeCl3/ITO film with enzyme (Mag. 50 KX)  

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Fig.4 (e) E-DAX of PANI/FeCl3/ITO film

Fig.4 (f) E-DAX shows composition of PANI/FeCl3/ITO film

Fig. 5 Current verses potential curves for the AChE/PANI/FeCl3/ITO sensor in 0.1M PBS

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Fig. 6 Effect of pH on the AChE/PANI/FeCl3/ITO electrode response of paraoxon

Fig. 7 Current response curve of (AChE/PANI/FeCl3/ITO) electrode 

Fig.8 Michaelis–Menten constant (Km) for the AChE/PANI/FeCl3/ITO sensor in 0.1M PBS

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Fig.9 Effect of inhibition time on paraoxon (in 0.5 µML−1) at 0.9 V vs. Reference electrode

Fig.10 Storage stability of the AChE/PANI/FeCl3/ITO sensor in 0.1M PBS

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