A newly emerging trend of chitosan-based sensing platform for the organophosphate pesticide detection using Acetylcholinesterase- a review

Accepted Manuscript A newly emerging trend of chitosan-based sensing platform for the organophosphate pesticide detection using Acetylcholinesterase-a review Hitika Patel, Deepak Rawtani, Y.K. Agrawal PII:

S0924-2244(18)30293-0

DOI:

https://doi.org/10.1016/j.tifs.2019.01.007

Reference:

TIFS 2393

To appear in:

Trends in Food Science & Technology

Received Date: 4 May 2018 Revised Date:

6 August 2018

Accepted Date: 8 January 2019

Please cite this article as: Patel, H., Rawtani, D., Agrawal, Y.K., A newly emerging trend of chitosanbased sensing platform for the organophosphate pesticide detection using Acetylcholinesterase-a review, Trends in Food Science & Technology, https://doi.org/10.1016/j.tifs.2019.01.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 2

A newly emerging trend of chitosan-based sensing platform for the organophosphate pesticide detection using Acetylcholinesterase- a review

3

Hitika Patela, Deepak Rawtania*, Y.K. Agrawala

4

a

5

Police Bhavan, Gandhinagar, Gujarat, India

ACCEPTED MANUSCRIPT

Institute of Research and Development, Gujarat Forensic Sciences University, Sector 9, Near

7

*Corresponding author:

8

Dr. Deepak Rawtani

9

Associate. Professor. (Nanotechnology), Institute of Research & Development,

11

Gujarat Forensic Sciences University,

12

Sector 9, Near Police Bhavan,

13

Gandhinagar 382007

14

Gujarat, India

M AN U

SC

10

RI PT

6

15 16

Mobile:+91- 9408276489

17

Email Address of the corresponding author: [email protected]

18

Ms. Hitika patel

20

Email address: [email protected]

23 24 25 26 27 28 29 30 31 32 33 34 35

EP

22

AC C

21

TE D

19

Abstract:

37 38 39 40 41 42 43 44

Background: Organophosphate pesticides have MANUSCRIPT been extensively used to protect the agricultural ACCEPTED produce from being damaged by the pests while growing and the subsequent degradation in its quality. However, in the process of doing so, the pesticides and their degradation products, enter the soil and water and start accumulating in the food products. On the consumption of such pesticide infected food products, Acetylcholinesterase is inhibited, which can be potentially damaging to the central nervous system of human beings. Acetylcholinesterase plays a pivotal role in the orderly functioning of the nervous system and in case of its failure to do so; there is a plausibility of deteriorating health in the individual.

45 46 47 48 49

Scope and approach: This review gives an insight into the recent approaches towards the rapid sensing of the deleterious pesticides. Numerous sensing platforms, comprising of chitosan as the key element of the immobilization matrix for the subsequent binding of acetylcholinesterase have been highlighted in this study. Chitosan plays the decisive role by aiding in the maintenance of the activity of immobilized Acetylcholinesterase.

50 51 52 53 54 55

Key findings and conclusions: The acetylcholinesterase enzyme-inhibition based biosensors pave the way for a speedy and feasible detection of the organophosphate pesticides present in the food articles by bypassing the copious pre-treatments. They also carry the possibility to be used for the real-life sample analysis. Thus, various transducers have been used in combination with the biopolymer chitosan, to produce highly sensitive biosensors for the detection of even trace amounts of these pesticides efficiently.

56

Keywords: Organophosphate pesticides; Acetylcholinesterase; Chitosan; Enzyme; Biosensor

M AN U

SC

RI PT

36

57 58

62 63 64 65 66 67 68 69 70 71 72 73 74

EP

61

AC C

60

TE D

59

75

1. Introduction Owing to the lower persistence ofACCEPTED the organophosphorus pesticides and their higher potency against MANUSCRIPT the insects (Evtugyn et al.,1997) than the organochlorine insecticides like DDT, Aldrin and lindane, these have widely been used in the agricultural sector(Rawtani et al., 2018; Timur, S., & Telefoncu, A.,2004). For example, organophosphorus pesticide like Methomyl has been used on the stored products as well as the marketable fruits and vegetable crops as a pesticide since the late 1960s(McCarroll et al., 2000). Another such pesticide of Pirimiphos-methyl (O-[2-(diethylamino)6-methyl-4-pyrimidinyl] O, O- dimethyl phosphorothioate), is a contact and fumigant OP that is applied on a thick range of crops such as citrus, olives, vines, vegetables, cane, ornamentals and other fruits for controlling thrips, mites, whiteflies and aphids (Sibanda et al.,2011). Owing to its high efficacy, low bioaccumulation and narrow persistence in the surroundings, Phoxim has been greatly used in the field of agriculture (Bachmann, T. T., & Schmid, R. D.). Some of the reaction products formed in the transformation process are much more deleterious to the human health than the parent compound and can have serious health implications (Donarski et al., 1989) while moving up the food chain and to the human beings (Dyk et al., 2011). Albeit the use of all these pesticides is decreasing perpetually due to the acute toxicity that often leads to respiratory paralysis and subsequent death (Abad et al., 1998), they still represent the major class of pesticides being used in the developing countries. They cause serious mishaps to the human health as well as the aquatic ecosystem equilibrium as they contaminate food and water. The organic toxins enter the food chain or water bodies, directly or indirectly, and pose a threat to very existence of the human being by showing high potency to the enzymes in cholinergic synapses i.e. Acetylcholinesterases (AChE) (Abad et al., 1998). This is a pivotal enzyme responsible for the normal functioning of the central nervous system in humans (Aldridge, W. N., & Davison, A. N., 1953). Organophosphorus pesticides work by the irreversible modification in the hydroxyl groups on the serine molecule at the catalytic site. This subsequently inhibits the AChE enzyme (Boublik et al., 2002), restricts the breakdown of the transmitter choline and further prevents the nervous transmission. As a result, acetylcholine starts accumulating in the synapses and over stimulates their receptor that ultimately proves detrimental to the nervous system (Long et al., 2015).

103 104 105 106 107 108 109 110

The methods routinely carried out for the analysis of pesticides employ high-performance liquid chromatography (HPLC),gas chromatography (GC) or mass spectroscopy (MS) or often combinations like GC-MS or LC-MS/MS (Kim et al.,2007).These general methods, on one hand, can identify single compounds in a mixture composite and have detection limits accordant to the laws imposed for pesticide residues in the environment (Timur, S., & Telefoncu, A.,2004), while on the other hand, these require extensive pre-treatment of the sample, massive labour resources, preconcentration steps and are insufficient for real-life sample analysis (Kim et al.,2007;Schulze et al.,2002).

111 112 113 114 115 116 117 118 119 120 121 122 123

Thus, rapid, dependable and feasible analytical method for the determination of minute quantities of these pesticides becomes the utmost priority while preserving the human health, safeguard it and for real-time analysis of practical samples (Ristori et al., 1996).Enzyme-based inhibition biosensors provided gratifying results where the inhibition of the enzyme activity directed the presence of pesticides. A possible pesticide poisoning was detected by the extent of enzyme inhibition that led to a concentration decrease in the reaction product of the enzyme than the reported normal values (Timur, S., & Telefoncu, A.,2004; Ristori et al.,1996). The methods employed in the fabrication of the biosensor for immobilization of the enzyme constitute of adsorption, entrapment, covalent binding, cross-linking, etc. (Anitha et al., 2004). The physical adsorption method of the enzyme onto a surface is sustained by the weak van der Waal’s forces that pose several limitations (Skládal et al., 1996). Thus, covalent bonding is often employed with bifunctional cross-linkers like glutaraldehyde or albumin or chitosan, etc., which are lined on one side by biomolecules and on the opposite side by the activated groups such as amino, hydroxyl or carboxyl (Skládal et al., 1996). In

AC C

EP

TE D

M AN U

SC

RI PT

76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102

most of the cases cross-linking is achieved by means of glutaraldehyde that can prove to be harmful to the activity of the enzyme (Ristori et al., 1996). The major disadvantages of this technique thus MANUSCRIPT encompass blockage of the activeACCEPTED sites of the enzymes, denaturation of enzymes triggered by the cross-linking agents and enzyme leakage (Bernabei et al., 1991). The enzyme-based inhibition biosensors are incompetent in specifically detecting or quantifying the various pesticides as most of the organophosphorus pesticides inhibit cholinesterase activity and thus, only the screening for the plausible contamination of the given samples can be accomplished (Andreescu, S., & Marty, J.-L., 2006).

132 133 134 135 136 137 138 139 140 141 142 143

Chitosan being a natural biocompatible biopolymer serves to be a noteworthy matrix for the immobilization of enzymes. This can be attributed to its numerous attractive properties such as excellent adhesion, availability of chemical modifications, non-toxicity, great mechanical strength, feasibility and ease in handling (Jayakumar, R. et al., 2010). Moreover, being a highly basic polysaccharide, chitosan serves to stand out of the range of other naturally occurring polysaccharides that are either neutral or acidic by nature (Kumar, M. N. V. R., 2000). When using chitosan as the immobilization matrix for acetylcholinesterase, chitosan plays the decisive role by aiding in the maintenance of the activity of immobilized Acetylcholinesterase (Yuan, Y. et al., 2011). Such a non-toxic biopolymer can be easily incorporated for its use in detecting any deleterious substances, like the organophosphate pesticides present in any food article. Thus, such enzyme-based sensing platforms imbibing chitosan, provide an upper hand over all the other enzyme-based inhibition biosensors.

144 145 146 147 148 149

In this paper, the different methods of detection (as shown in Fig. 1), various transducers used in combination with chitosan (as shown in table 1) and the immobilization techniques used (as shown in Fig. 2) for AChE used in pesticide determination have been reviewed. The disparate methods of detection and their principles have been depicted alongside the content of pesticides at different stages of handling of a food product in fig. 1. Whereas fig. 2 depicts the transducers assigned to the different approaches employed for the immobilization of the enzyme.

150 151 152 153 154 155 156

A multifarious number of organophosphorus pesticides could be detected using the AChE enzyme immobilized to a matrix containing chitosan. The modes of detection were mainly colorimetric and electrochemical modes. It could be concluded that in the colorimetric method, the immobilized AChE displayed a greater catalytic effect on the hydrolysis and affinity towards its substrate of acetylthiocholine chloride. In addition, many of the electrodes modified with AChE that were irreversibly inhibited by organophosphorus pesticides could resume its activity after immersing in 4.0 mM pralidoxime iodide (Skládal, P., 1992).

157

2. Colorimetric method of detection

158 159 160 161 162 163 164 165 166 167 168 169 170 171 172

An accelerated, inexpensive (Martinez et al., 2007), sensitive, handy, disposable and user-friendly (Martinez et al., 2008; Hossain et al., 2009) bioactive dipstick paper-based sensor was developed by Badawy, M.E.I., & El-Aswad A.F., 2014. This aided in the rapid sensing of organophosphate and carbamate pesticides of Methomyl and Profenofos (Li et al., 2011). Following the colorimetric assay given by Ellman (Ellman et al., 1961), the assay strip was made up of a biopolymer i.e. chitosan gel. It acted as the entrapping agent which was immobilized onto a Canson paper (1 × 10 cm). This paper support was in turn crosslinked by glutaraldehyde to the AChE enzyme and 5, 5’dithiobis (2-nitrobenzoic) acid (DTNB). The detecting reagent used was Acetylthiocholine iodide (ATChI) (Ellman et al., 1961). The assay protocol basically involved immersing of the pesticide containing solution, acting as the sample, into the biosensing region of the paper. Thereafter, the substrate containing paper was dipped in the ATChI solution to initiate the AChE catalyzed hydrolysis of the specific substrate, when it was incubated at 37° C for 5 minutes and thereby producing a color change to yellow. The absence or the decrease in the yellow color intensity gave the levels of residual activity of AChE left after binding of its inhibitors that reduce its activity. Hence it directed the levels of inhibitors present in the solution (as shown in Fig. 3). Fig. 3

AC C

EP

TE D

M AN U

SC

RI PT

124 125 126 127 128 129 130 131

summarises the working of the sensing platform as the decrease in the intensity of the yellow color upon the addition of inhibitors. The underlying principle of the protocol was that choline and acetic ACCEPTED acid molecules were yielded on the degradationMANUSCRIPT of acetylcholine molecules by the AChE enzyme (Ellman et al., 1961). The level of toxicity of organophosphate and carbamate pesticides, the distinct inhibitors of AChE, depends on the intensity of the yellow color produced and thus the inhibition of AChE.

179 180 181 182 183 184 185 186 187

The maximal absorbance of TNB was thereafter taken at 412 nm (Ellman et al., 1961). It was found that chitosan concentration of about 2% was optimum and glucose was added in the form of a stabilizer to the immobilization membrane(Kavruk et al.,2013). The ???? values, which are a measure of the affinity of the enzyme for its inhibitor (White et al.,1968), were found to be 978.81 and 7.7mM for Methomyl and Profenofos, respectively. This proved that methomyl is more potent in inhibitingthe activity of AChE than Profenofos. The detection limits were found to be reasonable in the range of 6.16 × 10−4 mM and 0.27 mM for Methomyl and Profenos, respectively. Glutaraldehyde was used here as the crosslinking agent for chitosan and AChE, that helped stabilize the enzyme along with DTNB and glucose as a stabilizer (Kavruk et al., 2013).

188 189 190 191 192

The major advantages of the developed biosensor included the use of cheap, easily available and expendable paper (Martinez et al., 2008; Hossain et al., 2009). Such a portable biosensor could also ease the on-site detection phenomenon to test the samples in their real environment (Martinez et al., 2008). But the demanding role here is played by the mode of immobilization of the enzyme in determining the activity of the immobilized enzymes.

193 194 195 196

Ellman-based colorimetry is an irksome and tedious method but can be passably used for the determination of AChE activity and carbamate pesticide detection. Nevertheless, it can function sans any other instrumentation. It also offers a simple and rapid semi-quantitative evaluation of contamination by cholinesterase’s inhibitors (Li et al., 2011).

197

3. Electrochemical method of detection

198 199 200 201 202 203 204 205 206 207 208 209 210

The Electrochemical methods are based on either a potentiometric (Du et al.,2007b), amperometric (Simonian et al.,2005) or a conductometric mode (Hassani et al.,2017) for measurement. Enzymebased electrochemical biosensors have an upper hand against the conventional methods due to their good sensitivity, selectivity, miniature sizes and rapid response (Jaffrezic-Renault, N., 2001). Electrochemical methods have a lot many advantages such as high reliability, basic instruments, rapid results, ease of operation, high sensitivity and it’s compatibility with complex samples (Jaffrezic-Renault, N.,2001).Retaining the native catalytic activity on an efficient immobilization of AChE is one of the vital considerations while developing an electrochemical biosensor that can be used for practical applications. The electrode’s electrocatalytic activity to TCl, the movement of the molecules related to the enzymatic reaction taking place within the biofilm and the biosensor stability also play key roles (Du et al., 2007b). The highlight of electrochemical biosensors is their unique ability to produce a quantifiable digital signal by converting the catalysis signal with the help of microfabrication electronics (Du et al., 2007b).

211

3.1. Potentiometric method

212 213 214 215

The fundamental to a potentiometric measurement is the rise of a specific organic acid along the course of hydrolysis (Timur, S., & Telefoncu, A., 2004).The prolonged signal response and reduced substrate sensitivity impede a majority of the biosensors relying on measurements by the pH detection (Timur, S., & Telefoncu, A., 2004).

216 217 218 219 220 221

The potentiometric detection system developed by Timur, S., & Telefoncu, A., 2004 had an underlying principle of that of the inhibition of the activity of AChE due to the arresting properties of organophosphates of Malathion, Parathion-methyl and Methamidophos. The enzyme was immobilized on the surface of a pH electrode with the help of a chitosan membrane. The optimum conditions for the assay were provided by the borate buffer (2.5mM, pH 8.5) at 25°C for the biosensor systems based on chitosan. Chitosan-based AChE electrode was fabricated on the pH

AC C

EP

TE D

M AN U

SC

RI PT

173 174 175 176 177 178

electrode by the deposition of the chitosan-acetate solution (1%) and AChE (30U) mixture solution on the electrode. Subsequently, it was cross-linked with the counter ion for chitosan i.e. ACCEPTED MANUSCRIPT tripolyphosphate solution (2%, pH 8.2) (Zihniog et al.,1995). During the application in pesticide analysis, the reference was provided by the recorded response from the saturated substrate concentration and the sensor was then cleaned up. This was further immersed in the buffer containing the pesticide solution with stirring (60min.) and thereafter a buffer stream was used to remove the surplus of the pesticide solution. A change in the output voltage in the membrane activity of the enzyme corresponded to the change in concentration of the protons as a result of the enzymatic reaction on the glass-electrode surface. A direct correlation with the initial activity helped figure out the percentage of residual activity. The enzyme electrode was defined so as to obtain the Thermal and Operational Stabilities, optimum pH (pH 8.5), optimum temperature (25°C) and the effective substrate concentration (1.42mM of AChCl). The detection of organophosphates without any pre-concentration step, in both aqueous and organic media, sans the requirement of any trained personnel proved to be advantageous to the proposed portable biosensors. The pesticides were effectively detected up to an appreciable range of 0.1 to 100mM for parathion-methyl and Methamidophos and 0.6 to 600mM for Malathion. However, in the presence of higher concentrations of pesticides, only a partial regeneration of the enzymatic activity was possible (Zihniog et al., 1995).

240

3.2. Amperometric method

241 242 243 244

The detecting principle in amperometric measurements is the comparison of the initial activity of the enzyme to that of its residual activity after exposure to OP compounds for the measurement of pesticides. The enzyme activity is found to be decreased after an exposure to OP compounds depending on the concentration and the time of exposure (Sun et al.,2009).

245 246 247

Amperometric biosensors could be further classified on the nature of the immobilization matrix employed for Acetylcholinesterase (as shown in Fig. 4) and the method employed in their fabrication.

248 249 250

3.2.1. On the basis of matrix employed

251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269

Based on the change in catalytic activity of Acetylcholinesterase (AChE) enzyme on prompting by Captan, Carbosulfan, 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and pentachlorophenol (PCP), led to the development of a novel bio-analytical method by Nesakumar et al.,2016 (Nesakumar et al., 2016). This reviewed the sensitivity and efficiency of inhibition using Pt/ZnO/AChE/Chitosan bioelectrode. By employing cyclic voltammetry, the fabricated Pt/ZnO/AChE/Chitosan bioelectrode could detect Captan, Carbosulfan, TCDD and PCP in their presence, due to them inhibiting the immobilized AChE enzyme on the electrode. Captan could be henceforth demonstrated from the results obtained as the most effective pesticide in inhibiting AChE activity. The detection limits of 1, 1, 5 and 50 nM were obtained for TCDD, PCP, Carbosulfan and Captan, respectively. The results obtained suggested that the fabricated models were aptly graded so as to enable the detection of Captan, Carbosulfan, TCDD and PCP in practical samples. The baseline was that the calibrated models were highly accurate and precise in detecting Captan, Carbosulfan, TCDD and PCP in local tap water samples. The devised bio-electrode exhibited improved inhibition efficiency, great recovery, high sensitivity and comparatively low relative standard deviation. These together helped in the quantification of the pesticide residues in local tap water samples and thus their application in real samples could be plausible. The contemplated method could be easily spread out to other inhibitors specific to an enzyme for their applicability in fabricating sensors relying on enzyme inhibition. Moreover, they could be also used in the testing of naturally available clean samples, like tap and spring water (Nesakumar et al., 2016).

270

AC C

EP

3.2.1.1. Inorganic compound

TE D

M AN U

SC

RI PT

222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239

3.2.1.2. Nanomaterial

272 273

Nanomaterials used as an elementACCEPTED of the immobilization matrix can be further characterized on the MANUSCRIPT basis of their dimensions as shown in Fig. 5.

274

3.2.1.2.1. Inorganic Nanomaterial

275 276 277 278 279 280 281 282 283 284 285 286 287 288 289

An AChE biosensor based on its inhibition by pesticides was devised by Yang et al., 2005.They employed a nanoporous ZrO2/Chitosan composite film as the immobilization matrix for the determination of Phoxim, Malathion and Dimethoate as the standard compounds. The surface of the composite film was treated by coating four layers of polyelectrolyte (chitosan/polystyrensulfonate) on the glassy carbon electrode and thus the interference by the plausible interferents was negligible owing to the specificity of the enzyme. The material used was a beneficial amalgam of inorganic nanoparticles, ZrO2, and the organic polymer, chitosan. The superiority of the sensor to other biosensors was in terms of it being inexpensive, straightforward and comparatively quick in its fabrication. Gratifying results were obtained by applying the proposed biosensor on real vegetable samples containing pesticides. The biosensor can therefore be potentially used in figuring out the pesticides in real samples directly. The structure of ZrO2being nanoporous greatly helped augment the active surface area over the total geometrical area that was available for effective binding of the enzyme in the developed biosensor (Liu et al.,2010). Additionally, the usage of glutaraldehyde, that denatures the enzyme, could be circumvented by immobilizing the enzyme on the nanoporous ZrO2. This would be depended on its absorption potential (Yang et al.,2005).

290

3.2.1.2.2. Reduced Graphene Oxide with a Nanomaterial

291 292 293 294 295 296 297 298

When rGO is combined with nanomaterials, be it a nanocluster or a nanoparticle, the synergistic effect improved the conductivity of the sensors (Zhou et al., 2010). They provided an excellent conductivity, catalytic activity and biocompatibility by retaining the biological activity for biomolecule immobilization (Chen et al., 2016). They also offered a hydrophilic surface which facilitated the immobilization of AChE when fabricating the organophosphorus pesticide biosensor. Their high surface area increased the surface loading of AChE and they provided a favorable microenvironment for the enzyme to maintain its biological activity (Chen et al., 2016).rGO, when applied as a film, could improve the sensitivity of the sensor(Chen et al., 2016).

299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314

An amperometric biosensor based on immobilizing AChE on 3-carboxyphenylboronic/reduced graphene oxide–gold nanocomposites modified electrode was fabricated for its use in determining the organophosphorus and carbamate pesticides by Liu et al., 2011. The esterification between the boronic acid group of 3-carboxyphenylboronic and the glycosyl of Acetylcholinesterase (Matsumoto et al., 2009) helped establish a strong immobilization of the AChE enzyme with comparatively greater activity on the electrode. It could be marked from the results that rGO and AuNPs have a potential to promote the electron transfer, but it was also seen that the conductive property of rGO was not as satisfying as that of GNPs. In the presence of the substrate of acetylthiocholine chloride, the organophosphorus and carbamate pesticides could be resolved satisfactorily. Moreover, the fabricated biosensor had an appraisable repeatability, brief response time, acceptable linear range, decreased detection limits and immense stability. The disadvantage is that of the low selectivity of AChE as both organophosphate and carbamate pesticides can inhibit AChE activity. However, on the other hand, this novel biosensor could portray a huge potential for estimating the outright amount during pesticide analysis. It could be concluded that the devised biosensor was suitable in the unmasking of Chlorpyrifos, Malathion, Carbofuran and Isoprocarb (Liu et al., 2011).

315 316 317 318 319 320

Another novel, stable and ultrasensitive OPs biosensor planted by the direct electrodeposition of electrochemically reduced graphene oxide (ERGO)-Au nanoparticles (AuNPs)-β-cyclodextrin (βCD) and Prussian blue-chitosan (PB-CS) on glassy carbon electrode was fabricated by Zhao et al., 2015,that could efficiently immobilize AChE. Due to the effective catalytic oxidation of TCh (Song et al., 2011) and the relocation of the oxidation potential by PB-CS (Wang et al.,2014), this sensing platform showed an elevated sensitivity (Arduini et al.,2012). The selectivity and sensitivity of the

AC C

EP

TE D

M AN U

SC

RI PT

271

biosensor were enriched by the reversible binding interaction of β –CD with its substrate. This contributed to a hike in the enhancement of the substrate. Broad linear range, rapid electrochemical response, sound reproducibility and decreased MANUSCRIPT detection limit of 4.14 pg/ml and 1.15 pg/ml for ACCEPTED Malathion and Carbaryl, respectively, was portrayed by the biosensor. The sensor exhibited a strong potential in its application for the analysis of practical samples directly as it displayed a fair accuracy in the sensing of pesticides in real samples (Zhao et al., 2015).

327 328 329 330 331 332 333 334 335 336 337

Zhang et al., 2015 devised a contemporary, stable, reproducible and a sensitive electrochemical AChE biosensor, using Phoxim as the classic OP compound. This was based on reduced graphene oxide (rGO) and silver nanocluster (AgNC) modified on a glassy carbon electrode (GCE). The immobilization of the enzyme on the support surface was enhanced by using carboxylic chitosan. It aided in the maintenance of the appropriate enzyme activity and further promotes the shuttling of electrons between the modified electrode and the enzyme. The amperometric response was refined due to the thiocholine concentration on the electrode surface, which was made easier by the specific affinity of the Ag and the mercapto groups. The biosensor exhibited a comparatively wider linear range and could detect even the trace amounts of Phoxim as low as 81 pM. The sensor could thus serve in the enzyme inhibition analysis and seemed a promising tool in the analysis of practical samples directly (Zhang et al., 2015).

338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357

Yet another multi-layered immobilization matrix based highly stable, reliable, rapid and simple electrochemical Acetylcholinesterase (AChE) biosensor was devised by Cui et al., 2018. This was done merely by adsorption of the enzyme on chitosan (CS), TiO2 sol-gel and reduced graphene oxide (rGO). The sensor was fabricated to determine OP compounds and Dichlorvos was taken as the model compound. The synthesized matrix, denoted as CS@TiO2-CS/rGO, had a mesoporous nanostructure (2-50 nm diameter). It was mechanically stabilized by the incorporation of CS and electrodeposition of a CS layer into/on the TiO2 sol-gel. Mesoporous nanostructure improved the enzyme loading by providing it a large surface area and a favorable environment. The positively charged CS layer could also enhance the immobilization amount and stability of negatively charged AChE. This provided the desired orientation and conformation for the biosensor fabrication. To improve the sensitivity of the sensor; reduced graphene oxide (rGO) was introduced as anrGO film onto the GC electrode for the measurement via differential pulse voltammetry (DPV). The detection limit was discernible to be 29 nM (6.4 ppb) detected in only about 25 minutes and was found to be superior to other reported values. The linear range was also found to be broader than the other reported values. The biosensor had varied real-life applications and thus provided an uncomplicated platform for the organophosphates detection. The catalytic activity of the AChE immobilized CS@TiO2-CS/rGO/glassy carbon electrode to acetylthiocholine was significantly higher than in the absence of any one of the components in the matrix. Nonetheless, the introduction of the CS layer could plausibly decrease the conductivity of the biosensor and limit the permeation of TCl through the matrix (Cui et al., 2018).

358

3.2.1.2.3. Chitosan Black (CB)

359 360 361 362 363 364

CB is a nanomaterial that confers some valuable electrocatalytic properties. It helps provide the electrode surface with a low electron transfer resistance (Rct) and improves the electrochemical performances. This is probably due to its good conductivity, more specific surface area and a high number of defect sites (Arduini et al.,2015). The drop-casting method serves a rapid, feasible and accessible method for generating thin films on the relatively small substrates (Talarico et al.,2016). Drop casting is a facile approach that requires no special equipment (Liu et al., 2014).

365 366 367 368 369 370 371

Drop casting method involving a single step was used in the preparation of a biosensor by Talarico et al., 2016, with carbon black (CB), chitosan and AChE. The screen-printed electrode (SPE) was modified with this dispersion. Paraoxon in drinking water was detected at legally permissible limits by the use of this low-cost and feasible biosensor while obtaining adequate recovery values. AChE embedded in CB allowed for the fast production of an uncomplicated biosensor. While the habitation of chitosan evoked the making of a biocompatible dispersion by dodging the use of organic solvents. The agglomeration of CB prevented by chitosan led to subsequent avoidance of

AC C

EP

TE D

M AN U

SC

RI PT

321 322 323 324 325 326

the sonication assisted by ultra-sonication. The moderate condition henceforth provided, aided the direct addition of the enzyme to the dispersion for the subsequent drop casting technique. This was MANUSCRIPT brought about by modification ofACCEPTED the screen-printed electrode in one step. The novel technique of immobilizing AChE on the combination of chitosan and CB led to the development of a biosensor with enhanced performance and a low detection limit of 0.05 µg/L. The challenges posed by this biosensor were the sensitivity and the performance. These were particularly affected by the binding of the active form of the molecule as ascribed to the screen printing technique. This technique was unstable and had a higher cross-reactivity towards anions. These factors when combined, led to a decrease in the lifespan of the technique. This sensor provided a promising tool for drinking waters by supervising their pesticide levels (Talarico et al., 2016).

382

3.2.1.2.4. Nanofibers

383 384 385 386 387 388 389 390 391

El-Moghazy et al., 2016 opportunely fabricated a super-sensitive and highly reproducible biosensor comprising of electrospun chitosan/poly (vinyl alcohol) blend nanofibers as a matrix for the immobilization of genetically engineered AChE. This electrochemical biosensor could bring about a quick detection of the model compound of Pirimiphos-methyl found in olive oil, sans any extensive pre-treatment. This was accompanied by a very low detection limit of 0.2 nM, exhibiting a concentration that is much lower than the maximum residue limit (MRL) i.e. 164 nM. A satisfactory analytical behavior was observed along with appreciable operational and storage stabilities. Thus, the noteworthy potential of the sensor in environmental monitoring and the food industry could be witnessed (El-Moghazy et al., 2016).

392 393 394 395 396

The baseline is that nanofibers act as the helping hand in doubling the response of the sensor and provides a higher enzyme loading by virtue of its unique characteristics. These specific aspects include the large surface area (Bhardwaj, N., & Kundu, S. C.,2010), spatial makeup and great porosity (Bhardwaj, N., & Kundu, S. C.,2010). By lowering the diffusion resistance of the substrate, nanofibers also aid in enhancing the catalytic activity of the enzyme (Ye et al.,2005).

397

3.2.1.2.5. Bimetallic alloy nanowires

398 399 400 401 402 403 404 405 406 407

Bimetallic alloy nanomaterials exhibit colossal potentials for ultrasensitive testing of the electrochemical measurements (Xu et al.,2011). These can be attributed to their electron interaction and change in atomic configurations (Thanh et al.,2016). The admirable electrocatalytic property and conductivity enhance the electron transfer rate. These along with the chemical stability and easy functionalization prove to be an upper hand for the bimetallic nanomaterials over the singlecomponent Nanohybrids(Ye et al.,2016). Pd comprising bimetallic nanomaterials are noteworthy for the ease of availability and inexpensiveness of Pd (Janyasupab et al.,2013). Such nanomaterials take Cu as the second candidate to combine with Pt or Pd to upgrade their catalytic activity (Xu et al.,2012). Conclusively, the nanowire structures portray alluring properties like high conductivity, more surface-to-volume ratio and no aggregation of the particles (Song et al.,2017).

408 409 410 411 412 413 414 415 416 417

Palladium-copper nanowires (Pd-Cu NWs) cohabited by chitosan served to be an ultrasensitive sensing platform for the detection of Malathion as the model compound. The fundamental of this inexpensive and efficient electrochemical biosensor devised by Song et al., 2017, was the production of an irreversible oxidation peak due to the hydrolysis of acetylthiocholine chloride (ATCl) by AChE immobilized on the modified electrode. This led to a subsequent decrease in current by inhibition of enzyme activity on encountering the OP pesticides. The sensor could adequately be applied for the analysis of Malathion in commercial fruits and vegetables with a low detection limit of 1.5 ppt (4.5 pM). The technique proved to be comparatively accurate and precise with a wider linear range. Thus, it portrayed a future for the onsite testing of OPs in samples like fruits and vegetables in their real environments (Song et al., 2017).

418

3.2.1.2.6. Multiwall Carbon Nanotubes (MWNTs)

419 420

Carbon nanotubes (CNTs) are nanoscale wires that have a remarkable biocompatibility, high conductivity, greater chemical stability, high mechanical strength and modulus and the possibility

AC C

EP

TE D

M AN U

SC

RI PT

372 373 374 375 376 377 378 379 380 381

of the modification of its sidewalls (Zhang et al., 2009). CNTs thus provide with some intriguing properties that lead to the development of highly efficient electrochemical biosensors having ACCEPTED MANUSCRIPT enhanced sensitivity and improved performances (Schulze et al., 2002). The higher length-todiameter aspect ratios and surface-to-volume ratios allow for the functionalization of CNTs with any desired chemical species. This aids in boosting the biocompatibility of the tubes (Liu et al., 2008). Thus, CNTs can be used for the highly sensitive, precise, accelerated and automated analysis of the OP compounds by blocking the activity of the AChE enzyme (Liu et al., 2008).

428 429 430 431 432 433 434 435 436 437 438 439 440 441 442

Guo et al., 2015 concocted a portable pesticides residues detection instrument by integrating an amperometric Acetylcholinesterase (AChE) biosensor and an analog signal detecting circuit. The sensing platform was modified with tin oxide (SnO2) nanoparticles, chitosan and multiwalled carbon nanotubes nanocomposite. The signal detecting circuit could reduce the noise level accompanied by the extremely weak signal of the AChE biosensor. The gold electrode was modified by the Nafion/AChE/MWNTs-SnO2-CHIT/Au composite film for better electrocatalytic ability in the oxidation of thiocholine. When a parallel was drawn between the AChE on CHIT/Au and onto MWCNTs-SnO2-CHIT/Au, the current was found to increase. Hence, it could be concluded that the MWNTs and SnO2 enhanced the electron transfer, the electrochemical response and reform the surfaces’ microarchitecture. This precise and prompt instrument could resolve the pesticides residues rapidly on-site in fruits and vegetables, within 15 minutes. The sensor could also process, display and store the data automatically. The detection limit was found to be 100 ng/L. The profitable sensing platform promises the rapid detection of a trace amount of pesticides in the agricultural commodities. Also, it had a response time lesser than that of the optical Raman spectroscopy instrument (Guo et al., 2015).

443 444 445 446 447 448 449 450 451 452 453 454

The modification of the surface of glassy carbon electrode (GCE) with Aniline (AN) and multiwall carbon nanotubes (MWNTs), led to the development of a simple and sensitive electrochemical biosensor (Liu et al.,2005) by Sun et al., 2011. The subsequent immobilization of AChE onto the sensor helped resolve Dichlorvos used as the model OP compound. The produced matrix could build up the transfer of electrons at a reduced potential and thus increase the sensitivity of detection. Dichlorvos was reported to be of high acute toxicity and could lower the activity of the enzyme to its substrate by irreversibly inhibiting it. The dip in the peak current increased proportionally with the concentration of Dichlorvos. The highly reproducible biosensor had a detection limit of 10ng/L in conjunction with good linearity. Where on one hand, the sensor posed a future application in the monitoring of pesticides even in trace amounts in the environment and food articles, on the other hand, the selectivity of the enzyme and the demand of a miniaturization of the system became an objection(Sun et al., 2011).

455 456 457 458 459 460 461 462 463 464 465 466 467

A layer-by-layer self-assembly modification technique was used by Zhao et al., 2012to formulate an electrochemical biosensor. This was devised by the use of glassy carbon electrode (GCE) modified by multi-walled carbon nanotubes (MWNTs) and chitosan (CS) to detect Methomyl. The so formed matrix was used for the immobilization of the AChE enzyme, which could be blocked from leaking out by the matrix’s formation method. Thus, the long-range sensitivity of the biosensor could be enhanced. The brisk biosensor was an amalgam of the exceptional electroconductivity of the MWNTs and the biocompatibility of the CS towards the creation of a highly sensitive sensing platform. It exhibited low detection limits of 10-11 g/L for Methomyl in seawater. Moreover, the electrochemical biosensor portrayed excellent storage stability and responses but some distinct analytes distressed the activity of the enzyme. Subsequently, the preliminary separation in complex samples became a necessity. However, the proposed biosensor could be used as a model in the creation of an efficient tool for pesticide monitoring in food articles and the environment (Zhao et al., 2012).

468 469 470 471

An elementary biosensor using the hybrid of multiwall carbon nanotubes (MWNTs) and chitosan (MC) was crafted by Du et al., 2007c. The AChE enzyme was immobilized for the quantitative detection of Triazophos used as the model compound. The amperometric sensor produced was highly reproducible, precise and sensitive. It gave a detection limit of 0.01 uM. The composite’s

AC C

EP

TE D

M AN U

SC

RI PT

421 422 423 424 425 426 427

netted structure interfered with the movement of the enzyme out of the electrode. The accelerated responses combined with the low detections limits make this biosensor available for the detection of MANUSCRIPT potential enzyme inhibitors or anyACCEPTED lethal compounds or in the monitoring of the environment. The low selectivity of the enzyme and the need for the miniaturization of the system pose some major challenges to the fabricated biosensor (Du et al., 2007c).

477 478 479 480 481 482 483 484 485 486

Du et al., 2010 created a one-step blend of multiwalled carbon nanotube on the gold nanoparticles (MWCNTs-Au). The so presented nanocomposite offered a hasty and adept method for the creation of a hydrophilic bed to facilitate the attachment of the biomolecule. This subsequently led to the fabrication of an extremely stable biosensor for the detection of Malathion used as the model compound. The exemplary conductivity showed by the matrix led to an increase in the affinity of AChE towards acetylthiocholine and thus could raise the loading of the biomolecule. The nanocomposites could also effectively enhance the transfer of electrons at the electrode surface and the corresponding electrochemical responses. The so devised biosensor exhibited an acceptable stability and reproducibility with a detection limit of 0.6 ng/ml. This laid its way in the detection of inhibitors of AChE or other enzymes (Du et al., 2010).

487

3.2.1.2.7. Quantum Dots

488 489 490 491 492 493 494 495 496 497 498 499 500 501 502

Organophosphate pesticides were detected by Dong et al., 2013 using a cost-effective and quick AChE biosensor using methyl parathion as the model compound of enzyme inhibition. Here, ZnSe quantum dots were functionalized with mercaptophenyl boronic acid to produce F-ZnSe QDs, which were specifically bound to the AChE. An amplified dual signal could be produced because of the excessive enzyme electrostatically immobilized on the graphene-chitosan nanocomposites (GRChi nanocomposites) cast previously upon the glassy carbon electrode. The nontoxic biocompatible F-ZnSe QDs were used as enzyme carriers. These not only allowed for a greater immobilization amount of the enzyme but also provided a favorable microenvironment to preserve the biological activity of the adhered enzyme to the fullest. The sensitivity along with the decent reproducibility of the sensing platform could be attributed to the incorporated GR-Chi nanocomposites that could possibly enhance the electron transfer rate on the electrode interface. The fabricated biosensor portrayed a detection limit of 0.2 nM and a comparatively better stability during storage. The sensor was successfully applied for the detection of methyl parathion in barbed soil and water samples. Thus, the proposed sensor paved its way for determining enzyme inhibitors and in the determination of organophosphorus pesticides (Dong et al., 2013).

503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522

Du et al., 2008a devised a novel, rapid and cost-effective technique using chitosan microspheres to recast glassy carbon electrode with CdTe quantum dots (QDs) and gold nanoparticles (GNPs) for immobilizing AChE. The highly conductive gold nanoparticles produced a synergistic effect on combining with AChE to enhance the affinity towards its substrate. This developed a highly sensitive, precise, reproducible and accurate AChE biosensor that used Monocrotophos as the model compound. The combined effect of CdTe-QDs and GNPs gave a copious number of advantages. For example, the combination could speed up electron transfer and subsequently catalyze the electro-oxidation of thiocholine. This had an impact on the detection sensitivity which was found to be 0.3 ng/ml. CdTe-QDs and GNPs together could also rule out the possibility of the enzyme leaking out of the electrode surface because of the covalent interactions between amino groups in AChE and carboxyl groups of QDs to produce Schiff bases. The fabricated sensor could hence be used in the analysis of garlic samples because of its permissible acceptability and provided a promising tool for the analysis of real-time samples. The sensor, however, suffered from certain disadvantages like the increase in the interfacial resistance after AChE immobilization because of the increase in the thickness of the interface. It also exhibited a low selectivity which could be attributed to the fact that AChE enzyme was inhibited to the same extent. The major drawbacks of the sensor are the fluorescence properties of the CdSe or CdTe QDs. When these are used as transducers, the process becomes essentially costly, time-consuming and arduous. The intensity of the fluorescent compounds keeps on fluctuating and is depended upon the analyte quantity. Moreover, the reaction needs special conditions like high temperature. Safeguarding the fluorescent

AC C

EP

TE D

M AN U

SC

RI PT

472 473 474 475 476

523 524

properties of the synthesized compound becomes a concern. A considerable number of coatings on QDs are required to preserve these properties (Du et al., 2008a).

525 526 527 528 529 530 531 532 533 534 535 536 537 538

In these biosensors, the quantum Dots used as immobilization support for AChE enzyme are impaired by their large size (10 to 30nm) (Bruchez et al., 1998) and their fluctuating behavior while measuring their fluorescence over long periods. During such long time intervals, analysis can be interrupted by the emissions (Nirmal et al., 1996). Albeit they have suffered certain demerits, they provide certain electronic and optical properties that make them unique for the use of AChE immobilization support (Du et al., 2008a). Such properties are conferred due to the very strong absorption cross sections and the emission energies that have narrow emission bands and are sizedependent (Peng et al., 1997). QDs also show broad excitation wavelengths when they are dimensionally similar to the immobilized biological material. Moreover, QDs exhibit a plethora of size-dependent properties (Jie et al.,2007), are superior to the organic fluorophores when their durability towards photo-bleaching is considered (Jaiswal et al.,2002) and can perform single molecules detection too (Liu et al.,2008). Thus, the bottom line is that the synergistic effect of the above properties of QDs increases the overall sensitivity of the electrochemical signal of the sensor (Du et al., 2008a).

539

3.2.1.2.8. AChE liposome bioreactor (ALB)

540 541 542 543 544 545 546 547 548 549

Taking Dichlorvos as the model compound, a highly specific, cost-effective and novel biosensor was fabricated by Guan et al., 2012 based on chitosan (CS) and AChE liposome bioreactor (ALB) multilayer films. The different layers of the multilayer films were assembled by immersing the glassy carbon electrode (GCE) in CS and ALB solutions alternately. The CS membrane used as the material for a carrier absorbed more ALB. CS and ALB had a synergistic effect on the copolymer network that could provide low detection limits of 0.86 ± 0.098 ug/L. The proposed biosensor was developed by a self-assembly technique and thus could exhibit high reproducibility, respectable stability and negligible interference of the electroactive substances. The biosensor so obtained had potential applications in the detection of trace organophosphorus pesticides as well as in the analysis of enzyme inhibitors (Guan et al., 2012).

550 551 552 553 554 555 556 557 558 559 560 561 562

Yan et al., 2013 constructed a multilayer films based novel, cheap and simple biosensor imbibing multiwall carbon nanotubes (MWCNTs), chitosan (CS) and AChE liposomes bioreactor (ALB). The model compound was taken to be Dichlorvos. It was observed that using 6 alternate bilayers of MWCNTs and ALB, could modify the glassy carbon electrode (GCE) in a way to enhance the electron transfer and improve the electrocatalytic ability. This could subsequently lead to strong amperometric signals and thus a low detection limit of 0.68 ± 0.076 ug/L. The individual components of the biosensor conferred some advantages to the biosensor, either alone or in conjunction with the other component. For instance, CS used as the carrier material absorbed more of the MWCNTs onto ALB. Conjointly, CS and ALB together could improve the detection limit sensitivity by giving a higher oxidation peak current at a certain potential than the one that was previously reported. The biosensor could thus be used for direct analysis of the practical samples of organophosphorus pesticides due to its fair reproducibility, high accuracy, quick response and a fair stability (Yan et al., 2013).

563 564 565 566 567 568

The liposomes in the above biosensors were an asset to the fabricated biosensors as they provided biocompatible microenvironments (Vamvakaki et al., 2005). In addition, they portrayed an ability to control the enzyme catalysis by supervising its physicochemical properties (Yan et al., 2013). Subsequently, these lipid membranes embedded with porins could restrict the free transport of the enzyme but not that of the substrate. This was due to the size limitations and thus the enzyme could be well secured from leaking out of the sensing platform (Yan et al., 2013).

569

3.2.2. On the basis of the technique employed

570

3.2.2.1. One step electrochemical deposition

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Electrodeposition provides a method which proves to be relevant for the judicious deposition of films and subsequently control the thickness of the films due to the sol-gel formation (Deepa et al., ACCEPTED MANUSCRIPT 2003). A clear and hasty method is the one-step electrochemical co-deposition technique. The simplicity (Sergeyeva et al., 2007), limited experimental conditions and the efficient deposition of films that can help control their thickness are the major advantages of this method over the conventional methods of detection (Deepa et al.,2003).

577 578 579 580 581 582 583 584 585 586 587 588 589

An electrochemical method was devised by Du et al., 2008b based on the coating of a planar gold electrode by a solution of gold nanoparticles embedded in chitosan hydrogel. This one step chemisorption/desorption method proved to be highly precise, smooth and accelerated for the determination of organophosphates. This was effected by setting up a favorable environment for the enzyme binding Malathion. The organophosphate pesticide was detected by this facile approach with a marked detection limit of 0.1 nM. When a potential of -0.7V was applied, the acetylthiocholine so produced on the hydrolysis of thiocholine was chemisorbed on the surface of AuNPs present in the formed matrix. A reduction in the current peak was observed directing that the inhibitor concentration was inversely proportional to thiocholine. This highly sensitive biosensor could detect Malathion even in trace amounts and thus the detection limit was found to be appreciably low. The plausible challenges posed by this biosensor are the selectivity of the inhibitor since the inhibition amount on AChE by organophosphate pesticides is nearly similar (Du et al., 2008b).

590 591 592 593 594 595 596 597 598 599 600 601

Methyl parathion was taken as the model compound by Gong et al., 2009 for its detection by the devised novel CaCO3-chi composite film using one-step electrodeposition method. The nano-sized film was an adept method for the fabrication of a 3D network-like structure. This could provide a suitable biocompatible microenvironment for both the substrate diffusion of the enzyme and also the immobilization of enzyme. Thus, a subsequent lowering of the oxidation potential of thiocholine was accomplished. This highly sensitive and steady biosensor was a facile step to create a shift in pH that could induce the electrodeposition method. The sensor portrayed a satisfactory reproducibility and a decent stability because of the high surface-to-volume ratio and greater hydrophilicity of the nano-sizedCaCO3. This proved to be a reliable enzyme immobilization matrix as the CaCO3 could aggravate the activity of the enzyme. On the one hand where a notable detection limit of about 1ng/ml was found, on the other hand, the efficient enzyme immobilization to the solid electrode surface might pose a challenge (Gong et al., 2009).

602 603 604 605 606 607 608 609 610 611

Du et al., 2007a devised an elementary and rapid matrix based on in-situ gold nanoparticles (GNPs) plantedin chitosan hydrogel containing tetrachloroauric (III) acid. This amperometric detection of Malathion and Monocrotophos was done with 0.01 ug/mL and 0.001 ug/ml, respectively, being their detection limits. This one-step electrochemical deposition was aided by the passive nature of the GNPs that proved to be biocompatible and prevented enzyme leakage of the electrode. The so formed CHIT-GNPs film was apt for retaining the high enzyme activity. This could be attributed to the biocompatible microenvironment provided by the non-toxicity of chitosan (Wu et al., 2003) and GNPs and could rule out the possibility of any enzyme leakage from the matrix. The electrochemically deposited film opened a new promenade for the electrochemical models and a budding application for the amperometric designs (Du et al., 2007a).

612 613 614 615 616 617 618 619 620 621

An AChE biosensor based on the novel technique of using chitosan membrane and Prussian blue membranes for modifying the glassy carbon electrode (GCE) was fabricated to detect the organophosphate pesticides. The dual membrane layer in the model created by Sun, X., & Wang, X., 2010 provided additional benefits. For instance, the Prussian blue (PB) membrane could boost the electron transfer rate due to its conductive and catalytic properties(Du et al., 2007b).The CS carrier soaked up an enormous enzyme amount and thus the two membranes had a synergistic effect heading for the catalysis of AChE(Du et al., 2007b). This rapid and facile amperometric biosensor could detect even low amounts of the pesticide such as 2.5 ng/l of Dichlorvos, 15 ng/l of Omethoate, 5 ng/l of Trichlorfon and 10 ng/l of Phoxim with a decent reproducibility and stability. This low-cost device thus gave different detection sensitivities in response to the disparate OP

AC C

EP

TE D

M AN U

SC

RI PT

571 572 573 574 575 576

622 623

compounds. Thus, the tremendous future potentials of the fabricated biosensor were portrayed (Sun, X., & Wang, X., 2010).

624 625 626 627 628 629 630 631 632 633 634 635 636 637 638

A chitosan-Prussian blue-multiwall carbon nanotubes-hollow gold nanospheres (Chit-PB-MWNTsHGNs) film was formulated by Zhai et al., 2013. Here, a modification was achieved of AChE and Nafion onto the film created by one-step electro co-deposition method on the gold electrode. The film of Chit-PB conferred a magnificent biocompatibility (Song et al., 2011) and thus could detect the model compounds of Malathion, Chlorpyrifos, Monocrotophos and Carbofuran with very low detection limits. The HGNs could speed up the transfer of electrons and subsequently this produced larger currents, greater selectivity and better stability. The embodiment of MWNTs and HGNs into Chit-PB hybrid film led to enhanced electron transfer reactions, the AChE catalytic activity and consequently remodelled the surface of the electrode (Che et al., 2011). Lastly, the Nafion films that were for the enzyme immobilization, saved it by providing a suitable microenvironment to conserve its activity. Thus, the anti-interference ability of the AChE/Chit-PB-MWNTs-HGNs/Au biosensor was enhanced. The collegial nature of Chit, PB, MWNTs and HGNs, led to the production of a highly accurate, sensitive and precise biosensor. Furthermore, this extensively stable and reproducible biosensor paved the way for a promising tool in the detection of pesticides by the analysis of practical samples directly (Zhai et al., 2013).

639

3.2.1.2. Flow- Injection Analysis (FIA)

640 641 642 643 644 645 646 647 648 649

A sensitive, fast, cheap, reproducible, stable, miniaturized and automatable flow injection detection of an organophosphorus insecticide by a novel biosensor was developed. This was by using AChE covalently bonded to chitosan cross-linked with multiwall carbon nanotubes (MWNT). The detection sensitivity of the sensor by Kandimalla, V.B., & Ju, H., 2006 could be enhanced by the MWNTs as they could bring about a reduction in the working potential for catalyzing the electrooxidation of thiocholine. The nanotube composite of chitosan–multiwall carbon, used as the immobilization matrix of AChE favoured loading as well as the covalent bonding of AChE to the matrix. Thus, the so formed fluid structure, consisting of uniformly distributed MWNTs, led to the attachment of the substrate and the enzyme. The sensor exhibited a rapid response with high precision and accuracy (Kandimalla, V.B., & Ju, H., 2006).

650 651 652 653

The use of flow-injection analysis in biosensors could provide a plethora of advantages like possible regulation of the reagents added at every step in the process, grading the enzyme activity, high sensitivity, low detection limits, quick responses that are automated and a recovery of the immobilized enzyme for future use (Evtugyn et al., 1997).

654

Conclusions

655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672

The baseline is that chitosan when used in conjunction to form an immobilization matrix for acetylcholinesterase, paved way for the development of distinct biosensors. The material used along with chitosan confers specific properties to the disparate biosensors that may prove advantageous in some or the other aspects. These sensing platforms provide for a cost-effective, rapid and dependable technique for pesticide detection. The biosensors are highly sensitive, precise, accelerated and automated analysis of the OP compounds by blocking the activity of the AChE enzyme can be accomplished. Further, they display a wider linear range, low detection limits and can detect even trace amounts of the pesticide. These sensors exhibit a strong potential in its application for the analysis of practical samples directly as they display a fair accuracy in the detection of pesticides in real samples. Thus, they portray a future for the onsite testing of OPs in samples like fruits and vegetables in their real environments. However, in these biosensing platforms, the selectivity of the enzyme and the demand for a miniaturization of the system became an objection. The challenges posed by the sensors are the selectivity of the inhibitor since the inhibition amount of AChE by organophosphate pesticides is nearly similar. When some of the factors involving the formation of the immobilization matrix are combined, they lead to a decrease in the lifespan of the technique. Thus, these enzyme-based inhibition biosensors are incompetent in distinctly detecting or measuring the amounts of the various pesticides. And hence, only the screening for the possible contamination of the given samples can be accomplished.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

When a comparison was drawn, it was found that amperometric biosensor provided an upper hand over the potentiometric sensors. For example, amperometric biosensors were quite simple, rapid ACCEPTED MANUSCRIPT and more sensitive than the potentiometric ones (Sun et al., 2009). Moreover, amperometric sensors provided a linear relationship depended on the concentration of analyte whereas the potentiometric sensors provided a logarithmic output signal (Ghindilis et al., 1998). To substantiate for the simplicity of the amperometric sensors, even a simple noble metal wire could be employed as the physicochemical transducer (Pohanka, M., 2009). However, if field analysis is to be considered, potentiometric sensors are simpler and suitable for such real-time analysis. Thus, it could be concluded that AChE biosensors based on amperometry, showed decent outcomes for analysis of pesticides wherein the enzymatic assay was employed as an indicator for measuring the organophosphorus compounds quantitatively (Pohanka, M., 2009).These acetylcholinesteraseinhibition based biosensors could be effectively applied to the real-time sample analysis and thus hold a great potential for their future use in the detection of even trace amounts of the pesticides.

686 687 688

Funding: This work has been financially supported by Gujarat Environment Management Institute, Gandhinagar, Gujarat, India (GEMI/726/1000/2017).

RI PT

673 674 675 676 677 678 679 680 681 682 683 684 685

SC

689 690

M AN U

691 692 693 694 695

699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718

EP

698

AC C

697

TE D

696

References:

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

1. Abad, J. M., Pariente, F., Hernández, L., Abruña, H. D., & Lorenzo, E. (1998). Determination of Organophosphorus and Carbamate Pesticides Using a Piezoelectric Biosensor. Analytical Chemistry, 70(14), 2848–2855. 2. Aldridge, W. N., & Davison, A. N. (1953). The mechanism of inhibition of cholinesterases by organophosphorus compounds. Biochemical Journal, 55(5), 763–766. 3. Andreescu, S., & Marty, J.-L. (2006). Twenty years research in cholinesterase biosensors: From basic research to practical applications. Biomolecular Engineering, 23(1), 1–15. 4. Anitha, K., Mohan, S. V., & Reddy, S. J. (2004). Development of acetylcholinesterase silica sol–gel immobilized biosensor—an application towards oxydemeton methyl detection. Biosensors and Bioelectronics, 20(4), 848–856. 5. Arduini, F., Di Nardo, F., Amine, A., Micheli, L., Palleschi, G.,& Moscone, D. (2012). Carbon Black-Modified Screen-Printed Electrodes as Electroanalytical Tools. Electroanalysis, 24,743–51. 6. Arduini, F., Forchielli, M., Amine, A., Neagu, D., Cacciotti, I.,& Nanni, F.(2015). Screenprinted biosensor modified with carbon black nanoparticles for the determination of paraoxon based on the inhibition of butyrylcholinesterase. Microchimica Acta, 182,643–51. 7. -Bachmann, T. T., & Schmid, R. D. (1999). A disposable multielectrode biosensor for rapid simultaneous detection of the insecticides paraoxon and carbofuran at high resolution. Analytica Chimica Acta, 401(1), 95–103. 8. Badawy, M.E.I., &El-Aswad A.F.(2014).Bioactive Paper Sensor Based on the Acetylcholinesterase for the Rapid Detection of Organophosphate and Carbamate Pesticides.International Journal of Analytical Chemistry, Article ID 536823, 8. 9. Bernabei, M., Cremisini, C., Mascini, M., &Palleschi, G. (1991). Determination of Organophosphorus and Carbamic Pesticides with a Choline and Acetylcholine Electrochemical Biosensor. Analytical Letters, 24(8), 1317–1331. 10. Bhardwaj, N., & Kundu, S. C. (2010). Electrospinning: A fascinating fiber fabrication technique. Biotechnology Advances, 28(3), 325–347. http://doi.org/https://doi.org/10.1016/j.biotechadv.2010.01.004 11. Boublik, Y., Saint-Aguet, P., Lougarre, A., Arnaud, M., Villatte, F., Estrada-Mondaca, S., & Fournier, D. (2002). Acetylcholinesterase engineering for detection of insecticide residues. Protein Engineering, Design and Selection, 15(1), 43–50. 12. Bruchez, M., Moronne, M., Gin, P., Weiss, S., &Alivisatos, A. P. (1998). Semiconductor Nanocrystals as Fluorescent Biological Labels. Science, 281(5385), 2013–2016. 13. Che, X., Yuan, R., Chai, Y., Li, J., Song, Z., Li, W., & Zhong, X. (2011). A glucose biosensor based on chitosan–Prussian blue–multiwall carbon nanotubes–hollow PtCo nanochains formed by one-step electrodeposition. Colloids and Surfaces B: Biointerfaces, 84(2), 454–461. 14. Chen, M., Hou, C., Huo, D., Dong, L., Yang, M., & Fa, H. (2016). A Novel Electrochemical Biosensor Based on Graphene and Cu Nanowires Hybrid Nanocomposites. Nano, 11(11), 1650128. 15. Cui, H.-F., Wu, W.-W., Li, M.-M., Song, X., Lv, Y., & Zhang, T.-T. (2018). A highly stable acetylcholinesterase biosensor based on chitosan-TiO2-graphene nanocomposites for detection of organophosphate pesticides. Biosensors and Bioelectronics, 99, 223–229. 16. Deepa, P. N., Kanungo, M., Claycomb, G., Sherwood, P. M. A., & Collinson, M. M. (2003). Electrochemically Deposited Sol−Gel-Derived Silicate Films as a Viable Alternative in Thin-Film Design. Analytical Chemistry, 75(20), 5399–5405. 17. Donarski, W. J., Dumas, D. P., Heitmeyer, D. P., Lewis, V. E., &Raushel, F. M. (1989). Structure-activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta. Biochemistry, 28(11), 4650–4655. 18. Dong, J., Zhao, H., Qiao, F., Liu, P., Wang, X., & Ai, S. (2013). Quantum dot immobilized acetylcholinesterase for the determination of organophosphate pesticides using graphenechitosan nanocomposite modified electrode. Anal. Methods, 5(11), 2866–2872.

AC C

719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773

EP

TE D

M AN U

SC

RI PT

19. Du, D., Chen, S., Song, D., Li, H., & Chen, X. (2008a). Development of acetylcholinesterase biosensor based on CdTe quantum dots/gold nanoparticles modified chitosan microspheres interface. Biosensors and Bioelectronics, 24(3), 475–479. ACCEPTED MANUSCRIPT 20. Du, D., Ding, J., Cai, J., & Zhang, A. (2007a). One-step electrochemically deposited interface of chitosan–gold nanoparticles for acetylcholinesterase biosensor design. Journal of Electroanalytical Chemistry, 605(1), 53–60. 21. Du, D., Ding, J., Tao, Y., & Chen, X. (2008b). Application of chemisorption/desorption process of thiocholine for pesticide detection based on acetylcholinesterase biosensor. Sensors and Actuators B: Chemical, 134(2), 908–912. 22. Du, D., Huang, X., Cai, J., & Zhang, A. (2007b). Amperometric detection of triazophos pesticide using acetylcholinesterase biosensor based on multiwall carbon nanotube–chitosan matrix. Sensors and Actuators B: Chemical, 127(2), 531–535. 23. Du, D., Huang, X., Cai, J., & Zhang, A. (2007c). Comparison of pesticide sensitivity by electrochemical test based on acetylcholinesterase biosensor. Biosensors and Bioelectronics, 23(2), 285–289. 24. Du, D., Wang, M., Cai, J., Qin, Y., & Zhang, A. (2010). One-step synthesis of multiwalled carbon nanotubes-gold nanocomposites for fabricating amperometric acetylcholinesterase biosensor. Sensors and Actuators B: Chemical, 143(2), 524–529. 25. Dyk, J. S. Van, &Pletschke, B. (2011). Review on the use of enzymes for the detection of organochlorine, organophosphate and carbamate pesticides in the environment. Chemosphere, 82(3), 291–307. 26. Ellman, G. L., Courtney, K. D., Andres, V., & Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7(2), 88–95. 27. El-Moghazy, A. Y., Soliman, E. A., Ibrahim, H. Z., Marty, J.-L., Istamboulie, G., &Noguer, T. (2016). Biosensor based on electrospun blended chitosan-poly (vinyl alcohol) nanofibrous enzymatically sensitized membranes for pirimiphos-methyl detection in olive oil. Talanta, 155, 258–264. 28. Evtugyn, G. A., Rizaeva, E. P., Stoikova, E. E., Latipova, V. Z., & Budnikov, H. C. (1997). The application of cholinesterase potantiometric biosensor for preliminary screening of the toxicity of waste waters. Electroanalysis, 9, 1124–1128. 29. Ghindilis, A. L., Atanasov, P., Wilkins, M., & Wilkins, E. (1998). Immunosensors: electrochemical sensing and other engineering approaches. Biosensors and Bioelectronics, 13(1), 113–131. 30. Gong, J., Liu, T., Song, D., Zhang, X., & Zhang, L. (2009). One-step fabrication of threedimensional porous calcium carbonate–chitosan composite film as the immobilization matrix of acetylcholinesterase and its biosensing on pesticide. Electrochemistry Communications, 11(10), 1873–1876. 31. Guan, H., Zhang, F., Yu, J., & Chi, D. (2012). The novel acetylcholinesterase biosensors based on liposome bioreactors–chitosan nanocomposite film for detection of organophosphates pesticides. Food Research International, 49(1), 15–21. 32. Guo, Y., Sun, X., Liu, X., Sun, X., Zhao, G., Chen, D., & Wang, X. (2015). A Miniaturized Portable Instrument for Rapid Determination Pesticides Residues in Vegetables and Fruits. IEEE Sensors Journal, 15(7), 4046–4052. 33. Hassani, S., Momtaz, S., Vakhshiteh, F., Maghsoudi, A. S,; Ganjali, M. R., Norouzi, P.,& Abdollahi, M. (2017). Biosensors and their applications in detection of organophosphorus pesticides in the environment. Archives of Toxicology, 91 (1), 109-130. 34. Hossain, S. M. Z., Luckham, R. E., McFadden, M. J., & Brennan, J. D. (2009). Reagentless Bidirectional Lateral Flow Bioactive Paper Sensors for Detection of Pesticides in Beverage and Food Samples. Analytical Chemistry, 81(21), 9055–9064. 35. Jaffrezic-Renault, N. (2001). New Trends in Biosensors for Organophosphorus Pesticides. Sensors, 1(2), 60–74. 36. Jaiswal, J. K., Mattoussi, H., Mauro, J. M., & Simon, S. M. (2002). Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nature Biotechnology, 21, 47. 37. Janyasupab, M., Liu, C.-W., Zhang, Y., Wang, K.-W., & Liu, C.-C. (2013). Bimetallic Pt–M (M=Cu, Ni, Pd, and Rh) nanoporous for H2O2 based amperometric biosensors. Sensors and Actuators B: Chemical, 179, 209–214.

AC C

774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830

EP

TE D

M AN U

SC

RI PT

38. Jie, G., Liu, B., Pan, H., Zhu, J.-J., & Chen, H.-Y. (2007). CdS Nanocrystal-Based Electrochemiluminescence Biosensor for the Detection of Low-Density Lipoprotein by Increasing Sensitivity withACCEPTED Gold Nanoparticle Amplification. Analytical Chemistry, 79(15), MANUSCRIPT 5574–5581. 39. Jayakumar, R., Menon, D., Manzoor, K., Nair, S. V, & Tamura, H. (2010). Biomedical applications of chitin and chitosan based nanomaterials—A short review. Carbohydrate Polymers, 82(2), 227–232. 40. Kandimalla, V.B.,& Ju, H. (2006). Binding of acetylcholinesterase to multiwall carbon nanotube-cross-linked chitosan composite for flow-injection amperometric detection of an organophosphorous insecticide. ChemistryEuropean Journal, 12, 1074–1080. 41. Kavruk,M., Özalp,V.C.,&Öktem, H.A.(2013).Portable Bioactive Paper-Based Sensor for Quantification of Pesticides. Journal of Analytical Methods in Chemistry, Article ID 932946, 8. 42. Kim, N., Park, I.-S., & Kim, D.-K. (2007). High-sensitivity detection for model organophosphorus and carbamate pesticide with quartz crystal microbalance-precipitation sensor. Biosensors and Bioelectronics, 22(8), 1593–1599. 43. Kumar, M. N. V. R. (2000). A review of chitin and chitosan applications. Reactive and Functional Polymers, 46(1), 1–27. 44. Li, H., Guo, J., Ping, H., Liu, L., Zhang, M., Guan, F., … Zhang, Q. (2011). Visual detection of organophosphorus pesticides represented by mathamidophos using Au nanoparticles as colorimetric probe. Talanta, 87, 93–99. 45. Liu, B., Yang, Y.-H., Wu, Z.-Y., Wang, H., Shen, G.-L., & Yu, R.-Q. (2005). A potentiometric acetylcholinesterase biosensor based on plasma-polymerized film. Sensors and Actuators B: Chemical, 104(2), 186–190. 46. Liu, S., Wang, J., Zeng, J., Ou, J., Li, Z., Liu, X., & Yang, S. (2010). “Green” electrochemical synthesis of Pt/graphene sheet nanocomposite film and its electrocatalytic property. Journal of Power Sources, 195(15), 4628–4633. 47. Liu, S., Yuan, L., Yue, X., Zheng, Z., & Tang, Z. (2008). Recent Advances in Nanosensors for Organophosphate Pesticide Detection. Advanced Powder Technology, 19(5), 419–441. 48. Liu, T., Su, H., Qu, X., Ju, P., Cui, L., & Ai, S. (2011). Acetylcholinesterase biosensor based on 3-carboxyphenylboronic acid/reduced graphene oxide–gold nanocomposites modified electrode for amperometric detection of organophosphorus and carbamate pesticides. Sensors and Actuators B: Chemical, 160(1), 1255–1261. 49. Liu, Y., Zhao, X., Cai, B., Pei, T., Tong, Y., Tang, Q., & Liu, Y. (2014). Controllable fabrication of oriented micro/nanowire arrays of dibenzo-tetrathiafulvalene by a multiple drop-casting method. Nanoscale, 6(3), 1323–1328. 50. Long, Q., Li, H., Zhang, Y., & Yao, S. (2015). Up conversion nanoparticle-based fluorescence resonance energy transfer assay for organophosphorus pesticides. Biosensors and Bioelectronics, 68, 168–174. 51. Martinez, A. W., Phillips, S. T., Carrilho, E., Thomas, S. W., Sindi, H., &Whitesides, G. M. (2008). Simple Telemedicine for Developing Regions: Camera Phones and Paper-Based Microfluidic Devices for Real-Time, Off-Site Diagnosis. Analytical Chemistry, 80(10), 3699–3707. 52. Martinez, A.W., Phillips, S.T.,Butte M.J.,& Whitesides, G.M.. (2007). Patterned paper as a platform for inexpensive, low-volume, portable bioassays.Angewandte Chemie— International Edition, vol.46 (8), 1318–1320. 53. Matsumoto, A., Sato, N., Kataoka, K., & Miyahara, Y. (2009). Noninvasive Sialic Acid Detection at Cell Membrane by Using Phenylboronic Acid Modified Self-Assembled Monolayer Gold Electrode. Journal of the American Chemical Society, 131(34), 12022– 12023. 54. McCarroll, L., Paton, M. G., Karunaratne, S. H. P. P., Jayasuryia, H. T. R., Kalpage, K. S. P., & Hemingway, J. (2000). Insecticides and mosquito-borne disease. Nature, 407, 961. 55. Nesakumar, N., Ramachandra, B.L., Sethuraman, S., Krishnan, U.M.,& Rayappan, J.B. (2016). Evaluation of Inhibition Efficiency for the Detection of Captan, 2,3,7,8Tetrachlorodibenzodioxin, Pentachlorophenol and Carbosulfan in Water: An Electrochemical Approach. Bulletin of Environmental Contamination and Toxicology, 96, 217-223.

AC C

831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887

EP

TE D

M AN U

SC

RI PT

56. Nirmal, M., Dabbousi, B. O., Bawendi, M. G., Macklin, J. J., Trautman, J. K., Harris, T. D., &Brus, L. E. (1996). Fluorescence intermittency in single cadmium selenide nanocrystals. Nature, 383, 802. ACCEPTED MANUSCRIPT 57. Peng, X., Schlamp, M. C., Kadavanich, A. V, &Alivisatos, A. P. (1997). Epitaxial Growth of Highly Luminescent CdSe/CdS Core/Shell Nanocrystals with Photostability and Electronic Accessibility. Journal of the American Chemical Society, 119(30), 7019–7029. 58. Pohanka, M. (2009). Cholinesterase based amperometric biosensors for assay of anticholinergic compounds. Interdisciplinary Toxicology, 2(2), 52-54. 59. Rawtani, D., Khatri, N., Tyagi, S., Pandey, G. (2018). Nanotechnology-based recent approaches for sensing and remediation of pesticides. Journal of Environmental Management, 206, 749-762. 60. Ristori, C., Carlo, C. Del, Martini, M., Barbaro, A., &Ancarani, A. (1996). Potentiometric detection of pesticides in water samples. Analytica Chimica Acta, 325(3), 151–160. 61. Schulze, H., Schmid, R., & Bachmann, T. (2002). Rapid detection of neurotoxic insecticides in food using disposable acetylcholinesterase-biosensors and simple solvent extraction. Analytical and Bioanalytical Chemistry, 372(2), 268-272. 62. Sergeyeva, T. A., Brovko, O. O., Piletska, E. V, Piletsky, S. A., Goncharova, L. A., Karabanova, L. V, … El’skaya, A. V. (2007). Porous molecularly imprinted polymer membranes and polymeric particles. Analytica Chimica Acta, 582(2), 311–319. 63. Sibanda, M. M., Focke, W. W., Labuschagne, F. J. W. J., Moyo, L., Nhlapo, N. S., Maity, A., … Brindley, G. W. A. (2011). Degradation of insecticides used for indoor spraying in malaria control and possible solutions. Malaria Journal, 10(1), 307. 64. Simonian, A. L., Good, T. A., Wang, S.-S., & Wild, J. R. (2005). Nanoparticle-based optical biosensors for the direct detection of organophosphate chemical warfare agents and pesticides. Analytica Chimica Acta, 534(1), 69–77. 65. Skládal, P. (1992). Detection of organophosphate and carbamate pesticides using disposable biosensors based on chemically modified electrodes and immobilized cholinesterase. Analytica Chimica Acta, 269(2), 281–287. 66. Skládal, P., Fiala, M., &Krejcí, J. (1996). Detection of Pesticides in the Environment Using Biosensors Based on Cholinesterases. International Journal of Environmental Analytical Chemistry, 65(1–4), 139–148. 67. Song, D., Li, Y., Lu, X., Sun, M., Liu, H., Yu, G., & Gao, F. (2017). Palladium-copper nanowires-based biosensor for the ultrasensitive detection of organophosphate pesticides. Analytica Chimica Acta, 982, 168–175. 68. Song, Y., Zhang, M., Wang, L., Wan, L., Xiao, X., Ye, S., & Wang, J. (2011). A novel biosensor based on acetylecholinesterase/prussian blue–chitosan modified electrode for detection of carbaryl pesticides. Electrochimica Acta, 56(21), 7267–7271. 69. Sun, X., & Wang, X. (2010). Acetylcholinesterase biosensor based on prussian bluemodified electrode for detecting organophosphorous pesticides. Biosensors and Bioelectronics, 25(12), 2611–2614. 70. Sun, X., Li, Q., & Wang, X. (2011). AChE biosensor based on aniline-MWNTs modified electrode for the detection of pesticides. In The 2011 IEEE/ICME International Conference on Complex Medical Engineering (pp. 441–444). 71. Sun, X., Wang, X. & Jia, C. (2009). Study on Carrier Material of Immobilization Acetylcholinesterase For Biosensor in Detection of Organophosphorus Pesticide Residues.IFIP International Federation for Information Processing, Volume 295, Computer and Computing Technologies in Agriculture II, Volume 3, eds. D. Li, Z. Chunjiang, (Boston: Springer), pp. 2283–2292. 72. Talarico, D., Arduini, F., Amine, A., Cacciotti,I., Moscone,D.,&Palleschi,G. (2016). Screenprinted electrode modified with carbon black and chitosan: a novel platform for acetylcholinesterase biosensor development. Analytical and Bioanalytical Chemistry, 408, 7299-7309. 73. Thanh, T. D., Balamurugan, J., Lee, S. H., Kim, N. H., & Lee, J. H. (2016). Novel porous gold-palladium nanoalloy network-supported graphene as an advanced catalyst for nonenzymatic hydrogen peroxide sensing. Biosensors and Bioelectronics, 85, 669–678.

AC C

888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942

EP

TE D

M AN U

SC

RI PT

74. Timur, S., &Telefoncu, A. (2004). Acetylcholinesterase (AChE) Electrodes Based on Gelatin and Chitosan Matrices for the Pesticide Detection. Artificial Cells, Blood Substitutes, and Biotechnology, 32(3), 427–442. ACCEPTED MANUSCRIPT 75. Vamvakaki, V., Fournier, D., &Chaniotakis, N. A. (2005). Fluorescence detection of enzymatic activity within a liposome based nano-biosensor. Biosensors and Bioelectronics, 21(2), 384–388. 76. Wang, B., Ji, X., Zhao, H., Wang, N., Li, X., Ni, R., & Liu, Y. (2014). An amperometric βglucan biosensor based on the immobilization of bi-enzyme on Prussian blue–chitosan and gold nanoparticles–chitosan nanocomposite films. Biosensors and Bioelectronics, 55, 113– 119. 77. White, A., Handler, P., Smith, E., &Stetten, D.. (1968). Principles of Biochemistry (2nd ed.). McGraw-Hill, (chapter 4). 78. Wu, L.-Q., Yi, H., Li, S., Rubloff, G. W., Bentley, W. E., Ghodssi, R., & Payne, G. F. (2003). Spatially Selective Deposition of a Reactive Polysaccharide Layer onto a Patterned Template. Langmuir, 19(3), 519–524. 79. Xu, C., Liu, Y., Su, F., Liu, A., &Qiu, H. (2011). Nanoporous PtAg and PtCu alloys with hollow ligaments for enhanced electrocatalysis and glucose biosensing. Biosensors and Bioelectronics, 27(1), 160–166. 80. Xu, C., Liu, Y., Wang, J., Geng, H., &Qiu, H. (2012). NanoporousPdCu alloy for formic acid electro-oxidation. Journal of Power Sources, 199, 124–131. 81. Yan, J., Guan, H., Yu, J., & Chi, D. (2013). Acetylcholinesterase biosensor based on assembly of multiwall carbon nanotubes onto liposome bioreactors for detection of organophosphates pesticides. Pesticide Biochemistry and Physiology, 105(3), 197–202. 82. Yang, Y., Guo, M., Yang, M., Wang, Z., Shen, G., & Yu, R. (2005). Determination of pesticides in vegetable samples using an acetylcholinesterase biosensor based on nanoparticles ZrO2/chitosan composite film. International Journal of Environmental Analytical Chemistry, 85(3), 163–175. 83. Ye, C., Wang, M.-Q., Zhong, X., Chen, S., Chai, Y., & Yuan, R. (2016). Highly sensitive electrochemiluminescenc assay of acetylcholinesterase activity based on dual biomarkers using Pd–Au nanowires as immobilization platform. Biosensors and Bioelectronics, 79, 34– 40. 84. Ye, P., Xu, Z.-K., Wang, Z.-G., Wu, J., Deng, H.-T., & Seta, P. (2005). Comparison of hydrolytic activities in aqueous and organic media for lipases immobilized on poly (acrylonitrile-co-maleic acid) ultrafiltration hollow fiber membrane. Journal of Molecular Catalysis B: Enzymatic, 32(4), 115–121. 85. Yuan, Y., Chesnutt, B. M., Haggard, W. O., & Bumgardner, J. D. (2011). Deacetylation of Chitosan: Material Characterization and in vitro Evaluation via Albumin Adsorption and Pre-Osteoblastic Cell Cultures. Materials, 4(8), 1399–1416. 86. Zhai, C., Sun, X., Zhao, W., Gong, Z., & Wang, X. (2013). Acetylcholinesterase biosensor based on chitosan/prussian blue/multiwall carbon nanotubes/hollow gold nanospheres nanocomposite film by one-stepelectrodeposition. Biosensors and Bioelectronics, 42, 124– 130. 87. Zhang, X., Guo, Q., &Cui,D.( 2009).Recent Advances in Nanotechnology Applied to Biosensors.Sensors, 9(2),1033-1053. 88. Zhang, Y., Liu, H., Yang, Z., Ji, S., Wang, J., Pang, P., … Yang, W. (2015). An acetylcholinesterase inhibition biosensor based on a reduced graphene oxide/silver nanocluster/chitosan nanocomposite for detection of organophosphorus pesticides. Anal. Methods, 7(15), 6213–6219. 89. Zhao, H., Ji, X., Wang, B., Wang, N., Li, X., Ni, R., & Ren, J. (2015). An ultra-sensitive acetylcholinesterase biosensor based on reduced graphene oxide-Au nanoparticles-βcyclodextrin/Prussian blue-chitosan nanocomposites for organophosphorus pesticides detection. Biosensors and Bioelectronics, 65, 23–30. 90. Zhao,Y.,Zhang, S.,Ma,J., Leng,KL., &Labuda, J.(2012). Determination of Carbamate Pesticide Methomyl Using Acetylcholinesterase / MWNTs-Chitosan Modified Glassy Carbon Electrode. Sensing in electroanalysis, 7,283−292.

AC C

943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997

998 999 1000 1001 1002 1003

91. Zhou, K., Zhu, Y., Yang, X., Luo, J., Li, C., & Luan, S. (2010). A novel hydrogen peroxide biosensor based on Au–graphene–HRP–chitosan biocomposites. Electrochimica Acta, 55(9), 3055–3060. ACCEPTED MANUSCRIPT 92. Zihniogˇlu, F., &Telefoncu, A. (1995). Diffusion characteristics of chitosan-entrapped microsomal UDP-glucuronyl transferase gel beads. Biochimica et Biophysica Acta (BBA) General Subjects, 1244(2), 291–294.

1004 1005

RI PT

1006 1007 1008

SC

1009 1010

M AN U

1011 1012 1013 1014

1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028

EP

1017

AC C

1016

TE D

1015

1029

ACCEPTED MANUSCRIPT

1030

Figure captions:

1032

Fig.1: Schematic representation for pesticide detection.

1033 1034

Fig. 2: Techniques employed for immobilization of Acetylcholinesterase on chitosan containing matrices.

1035

Fig.3: Reaction mechanism of acetylcholinesterase on organophosphate pesticides.

1036 1037

Fig.4: Classification of amperometric biosensors on the basis of matrix employed for Immobilization of acetylcholinesterase.

1038

Fig.5: Classification of Transducers on basis of techniques employed for immobilization.

RI PT

1031

1039

SC

1040 1041

M AN U

1042 1043 1044 1045 1046

1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063

EP

1049

AC C

1048

TE D

1047

1064

ACCEPTED MANUSCRIPT

1065 1066

Fig. 1

M AN U

SC

RI PT

1067

1068 1069

1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086

EP

1072

AC C

1071

TE D

1070

1087

ACCEPTED MANUSCRIPT

1088 1089

Fig. 2

1091 1092 1093 1094 1095 1096 1097 1098

AC C

EP

TE D

M AN U

SC

RI PT

1090

1099

ACCEPTED MANUSCRIPT

1100 1101 1102

Fig. 3

1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118

EP

1105

AC C

1104

TE D

M AN U

SC

RI PT

1103

1119

ACCEPTED MANUSCRIPT

1120 1121 1122

Fig. 4

1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138

EP

1125

AC C

1124

TE D

M AN U

SC

RI PT

1123

1139

ACCEPTED MANUSCRIPT

1140 1141 1142

Fig. 5

AC C

EP

TE D

M AN U

SC

RI PT

1143

1144 1145 1146 1147

Table 1:

ACCEPTED MANUSCRIPT

Table 1: Summary of the various modes of detection and transducers used

3.

Enzyme inhibitor

Colorimetric

CS/DTNB

Crosslinking

Methomyl,

6.16 × 10−4

(gluteraldehyde)

Profenofos

0.27mM

Ionic bonding

Malathion,

CS/pH electrode

Storage stability

References

NR

NR

Badawy, M.E.I., & El-Aswad A.F., 2014

0.6-600 mM

0.6–600mM

NR

Timur, S., & Telefoncu, A., 2004

Parathion- methyl,

0.1-100 mM

0.1–100mM

Methamidophos

0.1-100 mM

0.1–100mM

Malathion

0.1 nM

0.1-20 ng/ml

NR

Du et al., 2008a

Phoxim

0.081 nM

0.2-250 nM

Retained 92 % at 4° C after 30 days

Zhang et al., 2015

SC

M AN U

Potentiometric

Limit of Linearity range detection (LOD)

RI PT

Enzyme immobilization method

Amperometric (CV)

AChE-CSAuNPs/Au

Ionic bonding

Amperometric (CV)

AChE@CC S/AgNC/rG O/GCE

Crosslinking

Amperometric (CV)

AChECaCO3NPsCS/GCE

Diffusion

Methyl parathion

1 ng/ml

0.005-0.2 ug/ml

NR

Gong et al., 2009

Amperometric

AChE/CS@ TiO2CS/rGO/GC

Electrostatic interaction

Dichlorvos

29 nM

0.036 µM 22.6 µM

Wet storage in sterilized PBS at 4 °C and a dry

Cui et al.,2017

(DPV)

(carboxylic chitosan)

TE D

2.

Transducer

EP

1.

Mode of detection

AC C

Sr. No.

storage at −20 °C, respectiv ely, for 30 days

ACCEPTED MANUSCRIPT

AChECS/Pd-Cu NWs/GCE

Physical adsorption

Malathion

1.5 ppt

Amperometric (DPV)

CS/AChE/

Physical adsorption

Malathion,

4.14 pg/ml

Carbaryl

1.15 pg/ml

Pt/ZnO/AC hE/CS

Ionic bonding

Physical adsorption

Amperometric (CV)

AChE-MC/ GCE

Crosslinking (gluteraldehyde)

SC 1 nM

0.05–6 uM

Carbosulfan,

1 nM

10–30 uM

Song et al., 2017

After 28 days, a decrease of 8% when at 4° C

Zhao et al., 2015

NR

Nesakumar et al.,2016

5 nM

3–13 uM

50 nM

1–10 nM

Chlorpyrifos

100 ug/L

0.05–1.0 x105

NR

Guo et al.,2015

Triazophos

0.01 uM

0.03-7.8 uM

After 30 days, sensor retained

Du et al., 2007c

EP

Nafion/ACh E/MWNTsSnO2CS/Au

4.3-1.00 x 103 pg/ml

Maintain ed 91% at 4°C after 30 days

2,3,7,8tetrachlorodibenzodi oxin (TCDD), Pentachlorophenol (PCP)

AC C

Amperometric (CV)

7.98 - 2.00 x 103 pg/ml

Captan,

TE D

Amperometric (LSV)

M AN U

PB-CS /ERGO/Au NPs-βCD/GCE

5 -1000 ppt

RI PT

Amperometric (DPV)

7.8-32 uM

70 % of its initial current response

ACCEPTED MANUSCRIPT

AChECdte-GNPsCM/GCE

Covalent binding

Monochrotophos

0.3 ng/ml

Amperometric (CV)

AChEPB/GCE

Crosslinking (gluteraldehyde)

Dichlorvos, Omethoate, Trichlorfon

2.5ng/l

10ng/l-1ug/l

15ng/l

50n/l-10ug/l

SC 5ng/l

30 ng/l-5ug/l

10ng/l

50n/l-10ug/l

M AN U

Phoxim

1-1000 ng/ml

RI PT

Amperometric (CV)

After 30 days, it retained 92%

Du et al., 2008b

NR

Sun, X., & Wang, X., 2010

(MWCNTs/ ALB)6/ GCE

Ionic bonding

Dichlorvos

0.68 ± 0.076 ug/l

0.25-1.75 uM

NR

Yan et al., 2013

Amperometric (SEV)

(CS/ALB)5/ GCE

Ionic bonding

Dichlorvos

0.86 ± 0.098 ug/l

0.25-1.50 uM

After 15 days retained all its activity

Guan et al., 2012

Amperometric (CV)

AChE- ANMWNTs /GCE

Physical adsorption

10ng/L

50ng/l-1ug/l

NR

Sun et al., 2011

(AChE/CS)/ (MWNTs/C S)/GCE

Ionic bonding

Retained 80% of its original value after 21

Zhao et al., 2012

EP

AC C

Amperometric (CV)

TE D

Amperometric (CV)

Dichlorvos

Methomyl

50ug/l-5mg/l 10-11 g/l

10-10- 10-3 g/l

days

ACCEPTED MANUSCRIPT

94% of its initial current response when at 4° C after 14 days

Dong et al., 2013

1-1000 ng/ml

90% remainin g after 30 days at 4° C

Du et al., 2010

0.05 nM

0.05-75 nM

Zhai et al., 2013

Chlorpyrifos,

0.05 nM

0.05-75 nM

Monocrotophos,

0.1 M

0.1-50 nM

Carbofuran

2.5 nM

5-80 nM

The response current decrease d to 98.5% after 7 days whet at 4° C. after 30 days retained 90.2 %

1.0 nm

1.0–500 um

AChE/FZnSe/GRCS/GCE

Physical adsorption

Methyl parathion

0.2 nM

Amperometric (CV)

AChEMWCNTs AuCS/GCE

Physical adsorption

Malathion

0.6 ng/ml

Amperometric (DPV)

AChE/CSPBMWNTsHGNs/Au

Physical adsorption

Malathion,

0.5 nM - 0.5 µM

Amperometric (FIA)

AChE/CMC /GCE

AC C

EP

TE D

M AN U

SC

RI PT

Amperometric (CV, amperometry and chronoampero metry)

Covalent attachment (glutaraldehyde)

Sulfotep

Stored at Kandimalla, V.B.,& Ju, H., 2006 4°C under dry condition s, activity

Amperometric ( CV and chronoampero metry)

AChE/CS/C B-SPE

Physical adsorption

Amperometric (SEV)

AChE-CSGNPs/Au

Physical adsorption

Pirimiphos-methyl

0.2 nM

decrease d to 50% after 30 days. When at -20°C it retained 95% after 42 days and decrease d to 72% after 60 days

NR

When at 4°C,activ ity decrease d by less than 10% after 42 days

Elmoghazy et al., 2016

0.05 µg/l

0.1- 0.5 µg/l

after 1 week 85% activity was retained when at 4° C

Talarico et al., 2016

Malathion,

0.01 ug/ml

0.001-0.1 ug/ml

Du et al., 2007a

Monocrotophos

0.001 ug/ml

After 30 days storage,9 0% activity was

M AN U

Crosslinking (gluteraldehyde)

TE D

AChE/CSPVA NFM/SPE

Paraoxon

AC C

EP

Amperometric

SC

RI PT

ACCEPTED MANUSCRIPT

2-20 ug/ml

Phoxim,

-9

6.6x10-64.4x10 M

Dimethoate

1.7x10-6 M

1.0x10-8-5.9 x 10-7 M

Chlorpyrifos,

0.1 ppb

0.5–10 ppb

Malathion,

0.5 ppb

0.5–10 ppb

Carbofuran,

0.05 ppb

0.1–10 ppb

RI PT

5.0x10 M

Isoprocarb

0.5 ppb

NR

Yang et al.,2005

After 30day storage period, the sensor retained 90% of its initial current response when at 4° C

Liu et al.,2011

-4

Malathion,

SC

Covalent bonding (esterification)

1.3x10-6 M

M AN U

AChE/CPB A/AuNPs/r GOCS/GCE

Physical adsorption

TE D

Amperometric ( CV and chronoampero metry)

GC/ZrO2/C S/AChE

EP

Amperometric (CV)

AC C

48

retained when at 4° C

ACCEPTED MANUSCRIPT

2–10 ppb

ACCEPTED MANUSCRIPT Figure captions: Fig. 1: Schematic representation for pesticide detection. Fig. 2: Techniques employed for immobilization of Acetylcholinesterase on chitosan containing matrices Fig. 3: Reaction mechanism of acetylcholinesterase on organophosphate pesticides.

RI PT

Fig. 4: Classification of amperometric biosensors on the basis of matrix employed for Immobilization of acetylcholinesterase.

AC C

EP

TE D

M AN U

SC

Fig. 5: Classification of Transducers on basis of techniques employed for immobilization

ACCEPTED MANUSCRIPT Highlights:

EP

TE D

M AN U

SC

RI PT

Chitosan aids in maintaining the activity of immobilized Acetylcholinesterase. Potentiometric sensors are suitable for real-time analysis. Amperometric biosensors were simple, rapid and more sensitive. Detection of even trace amounts of the pesticides is possible.

AC C

• • • •