Sensing of Alphacypermethrin Pesticide Using Modified Electrode of Chitosan-Silver Nanowire Nanocomposite Langmuir Blodgett Film

ABSTRACT We report on the sensing of a hazardous pesticide alphacypermethrin (ACM) using electrochemical technique through a nanocomposite film modified electrode. The nanocomposite film comprising of octadecylamine, chitosan, polyvinyl alcohol-silver nanowires and haemoglobin, shortly (OCPAH), was prepared by Langmuir–Blodgett (LB) film deposition technique on various substrates and electrodes. The composite LB film was characterised by UV-visible spectroscopy and scanning electron microscopy, which confirms the stable and multilayer film. The LB film modified electrode was used for sensing ACM pesticide by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV) techniques. The achieved sensing parameters such as limit of detection as 14 nM, 5 nM and 10 nM; linear range as 10–100 nM, 10–40 nM and 50–100 nM/10–100 nM; and sensitivity as 0.418 µA/nM/cm2, 0.259 µA/nM/cm2 and 0.271 µA/nM/cm2 for CV, DPV and SWV techniques, respectively. The reported sensor is found to have stability of 74% upto 20 cycles, the relative standard deviation (RSD) value for metal ion/organic interference species as 2% and for real samples are within 1.4% using CV technique. The reported nanocomposite-based ACM pesticide sensor will open up new options for research on LB film nanocomposite-based sensing of different organophosphorus groups of pesticides.


Introduction
Pesticides are generally used in agriculture to kill or repel the plant attacking pests.Recent times, use of pesticide has increased exponentially to meet the food demand of the growing population [1].Farm use of pesticide is highest in tropical regions, and it is estimated that one-third of the global crop production currently depends on pesticide [2,3].There was an increase in agro production from 51 million tonnes (MT) (1950)(1951) to 252 MT (2016MT ( -2017) ) during the Indian green revolution, where pesticide played a major role [4,5].However, 0.1% of the applied pesticide reaches the target pest [6] and, remaining is contaminating water, environment and even food chain, causing neurological, immunological, respiratory and cancer [7].To counter pesticide problems, some strategies are evolving such as to minimise the use of synthetic pesticides, to maximise the use of biopesticide and to promote organic farming [8].Hence, it is essential to analyse pesticide quantitatively with a portable, low cost, high sensitive pesticide sensor.
There are conventional large size analytical instruments such as gas chromatographymass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) [6,9].However, these suffer from some disadvantages like time consuming, expensive, large size, high maintenance cost and require trained technicians, which limit their field application [10].The approximate cost of GC-MS and HPLC are around 39.5k USD and 14.9k USD, respectively [11].Hence, there is enough scope to develop a portable, sensitive pesticide sensor with the help of techniques such as electrochemical, optical, etc [12].Recent studies on enzyme-based and nanocomposite-based electrochemical sensors have shown higher sensitivity for detection of pesticide; however, it requires careful and innovative preparation of the composite matrices [13,14].
In this regard, the Langmuir-Blodgett (LB) method is a suitable tool for preparing uniform multilayer composite film on the electrode [15,16].Cyclic voltammetry (CV)based electrochemical tools can provide better stability of sensor [17], electron transfer kinetics [18], reversibility of a reaction [19] and a better calibration curve [20].The conducting nanomaterials in the sensing matrix such as carbon nanotube (CNT), gold nanoparticles (AuNPs), zirconium dioxide (ZrO 2 ), tin oxide (SnO 2 ), etc., have been used to improve the sensor performance due to their high surface area, catalytic and conducting properties [21][22][23][24].Chitosan have been selected as robust, biocompatible matrix for developing sensing platform due to its film-forming ability through its cationic amino group and chelating property to metal ions [25].Chitosan allows protein/enzymes to retain their conformation through its water bound molecules [26].Protein/enzymes have been used as primary recognition elements for pesticide sensors due to their redox and catalytic properties such as acetylcholinesterase (AChE), glucose oxidase, haemoglobin, etc [27][28][29].We have selected haemoglobin as redox-active protein in the composite film.Haemoglobin acts as redox protein in the composite and silver nanowires (AgNWs) help to improve current output of the sensor due to its higher surface area and catalytic property [30].
Alphacypermethrin (ACM) (MW 416.3 g/mol) has been selected as the model and target pesticide due to its versatile uses in agriculture and gardening [31].It is a pesticide belonging to the organophosphate group (OPs), which is structurally related to neurotoxic and consists of a phosphorus-oxygen (P¼O) or phosphorussulfer (P¼S) double bond [32].ACM is a mixture of two cypermethrin stereoisomers [33], which are used for a wide range of insect pests living in fruits, vegetables and tobacco such as Lepidoptera, Coleopteran, etc [34].ACM belongs to type II pyrethroid and is toxic to mammals, humans and aquatic environments [35].
We report on the sensing of ACM pesticide, which is largely ignored in the literature unlike its detection study by big size, costly instruments.A novel chitosan-silver nanowirebased nanocomposite LB film was prepared to modify a platinum (Pt) electrode.We achieved sensing parameters such as; limit of detection (LOD) as 14 nM, 10 nM and 5 nM; linear range (LR) as (10-100 nM, 10-100 nM and 10-40 nM/50-100 nM); and sensitivity as 0.418 µA/nM/cm 2 , 0.271 µA/nM/cm 2 and 0.259 µA/nM/cm 2 using CV, square wave voltammetry (SWV) and differential pulse voltammetry (DPV) techniques, respectively.The reported sensor is found to have stability of 74% upto 20 cycles, relative standard deviation (RSD) value for metal ion/organic interference as 2% and for real samples within 1.4% using CV technique.The results have been compared with other different existing detection methods on ACM pesticide.Our study explores research options for sensing study using LB nanocomposite for different kinds of pesticides.

Materials
Haemoglobin (Hb) stored below 4°C and octadecylamine (ODA) was purchased from Sigma Aldrich.The chloroform, chitosan (CS) (extra pure with viscosity 100 mPas), silver nitrate (AgNO 3 ) (extra pure AR), polyvinylpyrrolidone (PVP), ethylene glycol, sodium chloride (NaCl) and acetic acid were purchased from SRL, India.ACM was purchased from Indofil Industries Limited, India.Milli-Q deionised water with pH = 6.5 and resistivity = 18.5 MΩ × cm was used for LB experiment.The chemical structures of the constituent chemicals of the nanocomposite and pesticide analyte are presented in Figure 1.
The CS solution with a concentration of 0.1 M was prepared using acetic acid (0.1 M) solvent with continuous stirring for 48 hours.The Hb solution (0.05 mg/ml) was prepared by dissolving Hb in Milli-Q water.ODA solution (1 mM) was prepared by mixing ODA in chloroform solvent with stirring.

Preparation of silver nanowire
The AgNWs were prepared following the polyol process with a little bit of modification [30].PVP solution (50 mM) was prepared in 20 ml ethylene glycol solvent under stirring.AgNO 3 and NaCl were added to get the final concentration of AgNO 3 (3 mM) in 20 ml.The mixture was then heated using a kitchen grade LG microwave oven at 360 Watt for 3.5 minutes to obtain AgNWs.The final product was washed thoroughly with acetone to remove ethylene glycol and PVP with repeated filtering using filter paper (Whatman grade 602 H with pore size <2 µm) followed by washing with deionised water for multiple times.The sample was then centrifuged to remove unreacted PVP.

Cleaning of LB trough, substrate and electrode
The LB trough was filled with Milli-Q water and kept for 2 hours to remove any impurity and dust in the trough through the process of leaching.The trough and the barrier were cleaned by acetone, chloroform and tissue paper.The impurities present on the subphase were sucked by the aspirator pump.This process was performed several times to ensure that the water subphase was cleaned.All the substrates (glass, indium tin oxide (ITO), quartz, silicon wafer) were cleaned by ultrasonication along with soap solution, acetone and water.A uniform layer of water onto the slide confirmed the hydrophilicity of the slide.The electrodes (working, counter and reference) were sonicated for 5 minutes each with acetone, water and ethanol.It was kept in a desiccator for drying and for further experiment.

LB film preparation
A computerised LB film deposition instrument (model APEX LB-2007DC, Apex Instruments Co. India) was used for monolayer study and LB film preparation.The LB trough is made up of teflon and enclosed in a plexiglass box to reduce film contamination.A Wilhelmy-type balance (with accuracy of ±0.01 mN/m) was used to measure surface pressure on the water subphase of LB trough.The trough dimensions are width (130 mm), length (330 mm) and depth (20 mm).The deionised Milli-Q water was used to prepare the subphase.The pH and the resistivity of freshly prepared water were 6.8 and 18.2 MΩ × cm, respectively.All experiments were performed at a temperature of 20 ± 0.5°C.At least three independent runs were performed to check the reproducibility.
The nanocomposite LB film has been prepared using LB technique.The composite monolayer of OCPAH was prepared by spreading different solutions on a subphase using a micro syringe (Hamilton, Switzerland) within the surface pressure limit of 0.5 mN/m.The ODA was used as a floating lipid matrix, chitosan as a biocompatible polymer matrix, haemoglobin as electrochemically redox-active protein and AgNW nanocomponent as electron transfer enhancer [36][37][38].Initially, CS, PVP-AgNW and Hb solutions were spread, followed by the spreading of ODA to form a floating composite Langmuir monolayer.A reaction time of 30 minutes was given for stabilising the composite monolayer, and then the barrier was compressed at the rate of 5 mm/min.The pressure-area isotherm was recorded for each pure monolayer and composite monolayer.
The LB films were prepared by dipping the substrate or electrode into the water subphase before monolayer preparation.The film length and perimeter of the slide were kept as 19 mm and 60 mm, respectively.The monolayer was transferred to the substrate with lifting speed and dipping speed as 3 mm/min and 4 mm/min, respectively.The drying time for above and below the water subphase was kept as 5 minutes and 1 minute, respectively.The drying time after the 1st and 2nd layers was kept as 20 minutes so that the deposition of the first layer is strong on the substrate.
Surface pressure-time (π-t) kinetics of the monolayer was carried out by spreading the prepared solution on water subphase.The pressure-time measurements were recorded by the LB film deposition system, which gives the kinetics of the monolayer as a function of time.

Composite film characterisation
UV-visible spectroscopic characterisation was carried out on Hitachi Spectrometer (Model U-3900) in the absorption mode with wavelength range 200-800 nm, scan speed 300 nm/min, slit width 2 nm and optical path length 10 mm.The UV-visible spectra of nanocomposite LB film were recorded on a rectangular shape Quartz substrate.The fourier transform spectroscopic (FTIR) characterisation was done with the Bruker spectrometer (Model ALPHA) with OPUS 7.5 software on a silicon wafer substrate for powder and films.
Scanning electron microscopy (SEM) imaging of the composite films were carried out using SEM (JEOL, Model JSM-6360) for micrometre scale and FE-SEM (ZIESS, Model SIGMA 300) for nanometre scale.The nanocomposite LB film was deposited on a glass slide for SEM study.The operating voltage used was 20 KV.The film samples were mounted on brass stubs (30 mm diameter, 10 mm height) with the help of double side adhesive tape and gold conducting coating was made by Gold sputter (JFC 1100, JEOL) to increase conductivity and to protect nanocomposite film from the burning effect of electron beam.
Transmission electron microscopy (TEM) characterisation was done by JEOL TEM instrument (Model JEM-100 CX II) using Cu-coated carbon grid with a resolution of 1.4 A°, operating voltage 20-100 kV with 20 kV steps and magnification of 100,000-450,000×.The X-ray diffraction (XRD) characterisation was done using the Rigaku XRD instrument (Model TTRAX-III) using Cu-Kα X-ray with λ = 1.5406A° and scanning rate of 2°/minute.The XRD was used for AgNW characterisation.

Pesticide sensor characterisation
The platinum (Pt) working electrode was modified by LB film deposition technique.Briefly, the prepared solutions of ODA, Hb, PVP-AgNW and CS were spread on the LB subphase and a waiting time of 30 minutes was given to form a stable composite monolayer.The LB film was lifted using the mentioned LB parameter on the electrode and dried in a vacuum desiccator.
The CV, SWV and DPV measurements of LB film modified Pt electrode were carried on CH Instrument (Model CHI660D) with three electrode systems such as working electrode (green probe), counter electrode (red probe) and reference electrode (white probe).We used a glassy carbon electrode (GCE) as a counter electrode, silver/silver chloride (Ag/AgCl) as a reference and Pt as a working electrode.Initially, the CH instrument was given 15 minutes of warming time followed by a hardware test and selection of CV technique.The initial and final voltages were kept at −1 V and +1 V, respectively.The scan rate and sensitivity were optimised as 0.1 V/s and 5 × 10 −5 A/V, respectively.A KCl solution (0.1 M) was chosen as electrolyte and ACM pesticide was mixed with the electrolyte for sensing study in the concentration range of 10-100 nM.

Study of LB isotherm, pressure-area kinetics and UV-visible spectra of composite LB film
The composite monolayer was studied in the LB trough and subsequently transferred onto the substrate and electrode surface.Figure 2a shows the surface pressure-molecular area (π-A) isotherm of pure ODA, pure CS and the composite Langmuir monolayer.The lifting area/ molecule (nm 2 /molecule) of pure ODA and pure CS monolayer were found to be 0.12 and 0.2 nm 2 , respectively, which are in line with the earlier literature of ODA and CS monolayer [39,40].There is an appreciable shifting of the isotherm of composite monolayer compared to the characteristic lifting area of pure ODA and CS isotherms, which indicate the formation of composite [41,42].The reported OCPAH nanocomposite LB film is the first ever to be reported in the literature.
Figure 2b shows the pressure-time (P-t) kinetics study of the different composite monolayer after normalising the initial pressure value.It indicates the exponential decrease of surface pressure and final stabilisation within 30 min to 1 hour.The changes in the kinetics of composite monolayer than the pure monolayer also indicate the formation of composite monolayer [37,38].The kinetics have been fitted with double exponential decay equation (Equation 1) [43,44].The fitting parameters as well as the initial pressure of the decay kinetics are summarised in Table 1.
Where a 1 , a 2 and c are the parameters for reorganisation processes of the composite monolayer and τ 1 and τ 1 are the corresponding time constants.The values of a 1 and a 2 signify two components of the reorganisation process of monolayer; initially diffusion of the molecule and subsequent adsorption by air/water interface [44].This phenomenon was observed in the case of other protein monolayers such as haemoglobin, ovalbumin, etc [36,37].From Table 1, it is evident that the value of a 2 is higher and the value of τ 2 is lower, which indicates that the adsorption process is getting stronger with higher magnitude and faster with less time.It is interesting to note that there is negligible variation of the value of 'c', which is merely a mathematical constant and does not affect the reorganisation process of the monolayer [37].
Figure 2c shows the UV-visible spectra of OCPAH composite multilayer LB film with different numbers of layers (1, 5, 9, 12 and 17 layers), and Figure 2d shows the linear fitting of absorption intensity with layer numbers.The spectra contains a weak peak around 350 nm due to thin monolayer film, and it conforms to the presence of AgNW in the composite film [45].The correlation coefficient of linear fitting was achieved as R 2 = 0.93, which indicates that the layer by layer film was transferred linearly on the substrate without any loss of material.The peak at 350 nm corresponds to AgNW in nanocomposite film sample, while the absorption peak at 399 nm corresponds to AgNW in solution, and this shifting suggests the formation of composite of AgNW with other components in the film [46].

Characterisation of AgNWs
AgNWs have been used due to its high conductivity, high surface area due to wire shape as well as electron transfer catalytic property, which is expected to increase the current value in electrochemical study [47].The large surface area of AgNW provides greater chance to target analyte for reacting with the surface of nanowire, thereby increasing the sensitivity of the sensor.Also, silver is a highly active electrocatalyst in alkaline solutions that influence the kinetics of the reaction [48,49].
Figure 3a shows XRD peaks of AgNW corresponding to the planes (111), ( 200), ( 220), (311) and (222) which confirms the face-centred cubic (FCC) silver nanostructure as per the Joint Committee on Powder Diffraction Standards (JCPDS File No 04-0783) [45].Plane (111) has the maximum intensity and the calculated lattice constant for plane (111) to be 0.4084 nm, which is in agreement with the literature value of 0.4086 nm [45], and it indicates the high purity of FCC silver nanowire [50].Figure 3b shows the UV-visible spectra of AgNW in solution having peaks at 399 nm due to the plasmon excitation of electrons of silver nanowire [45].The shoulder peak at 350 nm is due to plasmon response of bulk silver, which is also observed generally for silver nanowire due to wire shape [46].
Figure 3c shows the TEM images of AgNW having noncircular tips, which have important consequences for sensing [51].The PVP used for AgNW preparation is acting as a reducing as well as capping agent.It may selectively bind to (100) facets and allow crystal growth along (111) facets as evidenced by its corresponding stronger XRD peak [52].The d-value was found to be 0.235 nm from TEM fringe pattern (Figure 3d) and 0.226 nm from TEM SAED pattern, which is in agreement with the literature value of 0.235 nm [53].

Characterisation of composite LB Film
Figure 4 shows the FTIR spectra of the composite film and its components chemicals in powder (Panel-I) and in film (Panel-II).It shows the characteristic IR peaks for chitosan, PVP and ODA as agreed from literature [54][55][56].The Table 2 shows the peak assignment of the FTIR spectra of the nanocomposite.The peak at 1526 cm −1 in the chitosan FTIR spectra is due to the N-H bending vibration bond and shifted to 1523 cm −1 due to Ag adsorption on nitrogen atoms after composite formation [54].The peak at 2884 cm −1 due to stretching vibration of C = O and shifted to the lower wavenumber 2850 cm −1 due to the interaction of Ag with the oxygen atom of PVP [57].
Figure 5 shows the SEM images of the nanocomposite LB film to search AgNW, Hb protein as well as to study the film morphology.Figure 5(a,b) shows SEM image in the micrometre scale, and Figure 5(c,d) shows FE-SEM in the nanometre scale showing the uniform film containing few aggregated Hb protein molecules along with the AgNW.The SEM image confirms the formation of uniform composite monolayer containing Hb protein on glass substrate (Figure 5d).This kind of protein LB monolayer SEM image was found in our earlier study also [38,41,42].The SEM and FE-SEM images were taken in two different SEM instruments just to come down to the nm range to get the protein aggregated image, and it became a complementary evidence also.The average diameter and length of the AgNWs were found to be 50 nm and 5 µm, respectively, as evident in the SEM image (Figure 5a) and TEM image (Figure 3a).

Determination of sensing parameters
ACM pesticide has been chosen as a model pesticide to carry out sensing study due to its toxicity and versatile uses for killing a wider range of pest in agriculture [33].In general, there are three major parameters of a sensor such as sensitivity, LOD and LR.In the electrochemical method of sensing, the calibration graph is derived from the changes of major peak current with concentration of the analyte i.e. pesticide.Pt electrodes have been chosen for electrode modification due to its higher current achieved in both bare and modified electrode (Figures S1, S2A). Figure 6a, b shows the CV response of the modified electrode with scan rate variation and its linear fitting.It is found that higher scan rate results in higher current, which may affect mass transfer of pesticide, and lower scan rate results in low current leading to lesser sensitivity with the same electrode.Hence, it needs to optimise to a moderate scan rate to accommodate both the issues and is decided optimised scan rate as 100 mV/s as per experimentation (Figure 6b) and as per reported scan rate for different pesticide sensors (20 mV/s, 50 mV/s, 100 mV/s) [61].The optimised scan rate (100 mV/s) has been fixed for all the necessary CV data such as, ACM sensing (10-100 µM), interference study, real sample analysis and repeatability.
The CV data of modified electrodes (Figure 6a) comprises anodic peak (E pa = 0.102 V), cathodic peak (E pc = −0.040V) at scan rate of 0.1 V/s with a peak-to-peak separation (ΔEp) of 0.155 V.The cathodic peak in case of Hb-ZrO 2 composite modified electrode was reported at −0.37 V, and the observed difference in the peak position may be due to the difference in composite material [62].The composite formation of Ag and Hb protein was also observed in our earlier work [38].The formal potential (E 0 ) is 0.077 V, which is the average of the cathodic and anodic peak [63].The linearity of peak current with scan rate or (scan rate) 1/2 indicates the electrochemical process to be diffusion controlled (Figure 6b, c) [64]; however, the shifting of peak current position indicates the adsorption controlled or double layer formation (Figure 6e) [65].In general, the slope value of the plot of logarithm of peak current vs scan rate can clarify the process to be purely diffusion controlled (slope = 0.5), purely adsorption controlled (slope = 1) or a mixture of diffusion and adsorption (slope in between 0.5 and 1) [66].The slope value of 0.66 (Figure 6d) together with cathodic peak shifting (Figure 6e), indicates the process to be adsorption and diffusion control [65,66].The anodic peak current to cathodic peak current ratio (i pa /i pc ) is found to be 1.26 i.e. greater than unity (1), which suggests the electrochemical process to be quasi-reversible process (Figure S3) [67].The electron transfer rate (Ks) is found to be 0.2082 ± 0.0032 s −1 using Laviron's method [68] (Equation 2) and intercept from Figure 6f.The surface coverage or surface concentration (Γ) of the active agent is found to be 2.212 × 10 −7 mol.cm −2 , using slope of Figure 6b and Equation 3 [64].Here, n is the number of electrons transferred, υ is the scan rate, i p is the peak current, F is the Faraday constant, R is the gas constant, T is the temperature, α is the transfer coefficient, and A is the area of the working electrode.
The sensor response with the amount of modifier needs to be standardised.In this direction, we have lifted three different layers of LB nanocomposite film on the Pt electrode and their CV response has been recorded.It shows the linearity in the cathodic peak current with the layer number (Figure S2b).It is also to mention that the amount of material (LB film layer) modified on the Pt electrode is difficult to measure.However, linear changes with the layer number certainly indicate the equal changes with the equal amount of electrode modifier.Also, the intrinsic technological advantage of LB trough allows us to precisely control the number of layers and subsequently amount of electrode modifier.Hence, LB film modified electrode sensors can be made standard and reliable with repeatable peak current response.
Figure 7 (a,c,e) shows the CV, DPV and SWV data of the modified electrode with varying concentration of ACM pesticide.Figure 7(b,d,f) shows the calibration curve of the reported sensor using CV, SWV and DPV techniques.The CV signal variation with pesticide concentration is less linear in anodic peak current (R 2 = 0.81) (Figure S4) in comparison to the cathodic peak current (R 2 = 0.98) (Figure 7b), which is evidenced in the R 2 value of the linearity fitting.Figure S5a shows the changes of cathodic peak (−0.040V) with ACM concentration.Figure 7b shows the CV calibration curve for the reported sensor, which is derived from cathodic peak current variation with ACM concentration.The sensitivity of the sensor has been calculated using slope of the calibration graph and the working area of the electrode (Equation 4) [69].The sensitivity of the reported sensor using CV, DPV and SWV was found to be 0.418 µA/nM/cm 2 , 0.259 µA/nM/cm 2 and 0.271 µA/nM/cm 2 , respectively, which is a single reported data till date on ACM pesticide using electrochemical techniques.The area of the Pt working electrode was calculated using slide callipers and was found to be 0.2826 cm 2 .It is to be noted that the area including PTFE coating is 0.3455 cm 2 and this entire area is being modified by LB method.However, PTFE is insulating in nature and the working area (0.2826 cm 2 ) will be functional only and is considered as the area of modified Pt electrode.The LOD has been calculated using slope and residual standard deviation (RSD) of the calibration curve (Equation 5) [70].The LOD using CV, DPV and SWV is found to be 14 nM, 5 nM and 10 nM, respectively, which is near the comparable detection limit of ACM pesticide by heavy instruments like GC-MS, TLC, HPLC and UV-visible spectroscopy [71][72][73][74].
The LR is the range of sensing parameters (such as concentration of pesticide), where the response signal differs by maximum 5% or the response signal is linearly proportional with tolerable R 2 value of ≥0.95 [70,75].The LR value of the reported sensor using CV, DPV and SWV techniques has been calculated by the linear fitting of the signal response with concentration of ACM pesticide (Figure 7b,d,f).The LR value is found as 10-100 nM (CV), 10-40 nM and 50-100 nM (DPV) and 10-100 nM (SWV) with R 2 value of the linear fitting as 0.98, 0.96 and 0.97 respectively.In DPV technique, the LR needed to be split into two ranges (R 2 = 0.96) to increase the R 2 value from the single range (R 2 = 0.85).
Figure S5b shows the CV stability data of the reported sensor upto 20 number of cycles.The peak current gradually changes in every cycle and attains a saturation.The increase in peak indicates the population of active sites increases on the Pt electrode and the decrease in peak current indicates the passivation of the surface of the electrode [76].The stability of the ACM sensor has been calculated using Equation 6 [76], and the stability value of 74% indicates that the signal response of the modified electrode to be stable.
The reproducibility and repeatability of the reported sensor have been carried out (Figure S6 a, b) using three numbers of LB film modified electrodes.It is found that RSD from three electrode responses is 1.5%, which represents the reported sensor to be reproducible.Figure S6 c, d shows a repeatability graph by the CV response for 20 cycles and their RSD is found to be 8%.This indicates the sensor developed by LB film modified electrode can give reproducible data as per the experimental data and also from the fact that the LB film thickness can be controlled by the layer number.
In literature, pesticide sensing applications have been carried out using different electrochemical methods such as CV, SWV and DPV [77,78].CV is widely used as it provides essential information, such as the process reversibility and types of redox processes present in the analysis (matrix, analyte and electrodes) [79].Whereas, the pulse voltammetry techniques such as DPV and SWV are very sensitive, often allowing direct analyses of analyte at the ppb (parts per billion) level and even the low ppt (parts per trillion) [80].DPV technique is comparably slow compared to CV [81].Kalinke et al. prepared a dopamine sensor using CV, DPV and SWV techniques and found the R 2 value of their calibration curve to be 0.98, 0.99 and 0.99, respectively [82,83].The reported sensor achieve higher sensitivity in CV (0.418 µA/nM/cm 2 ) than DPV (0.259 µA/nM/cm 2 ) and SWV (0.271 µA/nM/cm 2 ) and broader LR (10-100 nM).

Interference studies and real sample analysis
The interference studies of the reported ACM pesticide sensor have been carried out in presence of 2 µM metal ions (Zn 2+ , Cu 2+ , Ni 2+ , Pb 2+ , Al 3+ , Mg 2+ ) as well as 2 µM organic interfering species (ascorbic acid, aspartic acid, glutamic acid, citric acid, glycine) along with 15 nM of ACM pesticide (Figure 8a).It is found that the interfering species could change the CV response of LB film modified Pt electrode only upto a little extent.The overall RSD of the sensor peak current response in the presence of metal ions and organic interfering species are within 2%, which reveals the good consistency of sensor response in the presence of interfering agents (Figure 8a).Also, the RSD value in peak potential response in the presence of interfering species is found to be within 2.4%, which is a complementary evidence in the interference study (Figure S7).
The reported ACM pesticide sensors have been tested for real sample analysis in the case of chilli and tap water in the presence of 15 nM ACM pesticide (Figure 8b).The ACM pesticides have been widely used in chilli farming, and hence, it is used in real sample analysis.The real sample analysis data have been compared with standard distilled water results and found the RSD in case of chilli and tap water as 1.4% and 0.2%, respectively.
To the best of our knowledge, there are no literature reports on the ACM pesticide sensing by electrochemical method exclusively by using LB nanocomposite film unlike the large size instruments like GC-MS, HPLC.Table 3 shows the results of the reported ACM pesticide sensor as well as detection results from other heavy instruments.

ACM Pesticide interaction with nanocomposite
The nanocomposite used for electrode modification comprises haemoglobin, PVP-AgNW, octadecylamine and chitosan.Haemoglobin has been used due to its direct electron transfer capacity between Hb and electrode [84], which is wrapped by the suitable biocompatible chitosan microenvironments [85].The ACM-like organophosphate pesticides contain sulphur, phosphorus or nitrogen groups, which are easy to adsorb on the surface of AgNPs via covalent interaction [86].Also, the reducing group of −OH or −NH in ACM pesticides readily reacts with the oxygen-containing haemoglobin [87].
The interaction between nanocomposite and ACM pesticide has been explored experimentally using CV (Figure 9a, b) and UV-visible spectroscopy (Figure 9c, d).It is found that the cathodic peak position is shifting, and the peak current is decreasing with ACM concentration.Finally, the cathodic peak becomes flat in the µM range of ACM, which is the reflection of denaturation and destruction of haemoglobin due to µM concentration of ACM pesticide.The denaturation of haemoglobin is also evident in the UV-visible spectroscopic data by the abrupt shifting of the Soret band position in the µM range of ACM (Figure 9d) [37].In conclusion, the interaction of ACM existed with haemoglobin and may be with AgNW through adsorption [37].

Conclusion
An electrochemical pesticide sensor has been developed to detect a hazardous ACM pesticide using CV, DPV and SWV technique with sensitivity 0.418 µA/nM/cm 2 , 0.259 µA/ nM/cm 2 and 0.271 µA/nM/cm 2 using a novel chitosan-silver nanowire-based nanocomposite LB film modified electrode.To the best of our knowledge, the sensing study of the ACM pesticide has not yet been explored in literature using nanocomposite matrix unlike the big size costly instrument.The formation of the composite (OCPAH) was studied by the surface pressure isotherm using LB technique as well as SEM and FTIR technique.The achieved sensing parameters such as LOD as 14 nM, 10 nM and 5 nM; LR as (10-100 nM, 10-40 nM and 50-100 nM and 10-100 nM), respectively, for CV, DPV and SWV techniques.The CV showed the broad LR (10-100 nM) with R 2 = 0.98, high sensitivity (0.418 µA/nM/ cm 2 ) compared to SWV and DPV techniques.The results were found to be promising as

Figure 2 .
Figure 2. (a) Pressure-area isotherm of pure ODA, pure CS, ODA+CS and OCPAH composite monolayer.(b) Pressure-time kinetics data and their fitting data by double exponential decay equation (c) UVvisible spectra of composite LB film with different layer numbers (1-17 layer) (d) Peak absorbance (at 350 nm) with layer no and its linear fitting.

Figure 4 .
Figure 4. FTIR spectra of the nanocomposite and its different components in powder form (Panel-I) and in LB film (Panel-II).

Figure 5 .
Figure 5. (A-D) SEM images of the composite LB film at different resolution and magnification.

Figure 6 .
Figure 6.(a) CV response of LB film modified Pt electrode with scan rate variation.(b) Linear plot of cathodic peak currents vs scan rates.(c) Linear plot of cathodic peak currents vs square root of scan rates.(d) Linear plot of logarithmic cathodic peak currents vs logarithmic scan rates.(e) Linear plot of cathodic peak position vs scan rate.(f) Linear plot of anodic-cathodic peak separation vs ln (scan rate).

Figure 7 .
Figure 7. (a) CV data of composite LB film modified Pt electrode with concentration of pesticide in the nM range.(b) CV peak current vs. pesticide concentration and its linear fitting.(c) SWV data of composite LB film modified Pt electrode with concentration of pesticide in the nM range.(d) SWV peak current vs. pesticide concentration and its linear fitting.(e) DPV data of composite LB film modified Pt electrode with concentration of pesticide in the nM range.(f) Peak current vs. pesticide concentration and its linear fitting.

Figure 8 .
Figure 8. (A) Cathodic peak current of LB film modified Pt electrode in presence of different interfering agents and in presence of 15 nM ACM pesticide.(B) Comparison of cathodic peak current of LB film modified Pt electrode in ideal distilled water and in real samples (chilli, tap water) and in presence of 15 nM ACM pesticide.
Cyclic Voltammetry, DPV = Differential Pulse voltammetry, SWV = Square wave voltammetry, GC-MS = Gas chromatography-Mass Spectroscopy, TLC = Thin Layer Chromatography, HPLC = High-Performance Liquid Chromatography, NR = Not Recorded.compared with other existing methods.The present study opens up new research options on high sensitive sensor development for other different kinds of hazardous organophosphorus pesticide using LB film-based nanocomposite modified electrodes.

Figure 9 .
Figure 9. (a) CV (cathodic peak current) response of LB film modified Pt electrode with ACM concentration (nM to µM).(b) Changes in cathodic peak current and peak position with concentration of ACM.(c) Normalised UV-visible spectra of haemoglobin with concentration of ACM, (d) Change in haemoglobin Soret band position with concentration of ACM.

Table 1 .
Fitting Parameters of Pressures-Time (P-t) Data of Different Composite Monolayers by Double Exponential Decay Equation.

Table 2 .
Table for peak assignment of FTIR spectra of the nanocomposite.

Table 3 .
Sensing Data of the Reported ACM Sensor and Literature Data using Different Techniques.