organophosphate ( op ) compounds are used as pesticides and insecticides in agriculture and have found applications in military practices as chemical warfare agents .
commonly used op pesticides include parathion , malathion , methyl parathion , chlorpyrifos , diazinon , dichlorvos , phosmet , and fenitrothion . according to world health organization reports , there are three million pesticide poisonings that occurred worldwide every year ; most of them are op - related , and 200,000 deaths occur due to either self - poisoning or occupational exposure .
reports in the literature demonstrate that op pesticides affect the nontarget organisms such as birds and fish , as well as humans through exposure to water resources , fruits , vegetables , and processed foods contaminated by op pesticides .
the op pesticides irreversibly inhibit the activity of acetylcholine esterase , an enzyme essential for the proper functioning of the central nervous system in humans and insects , resulting in the accumulation of the neurotransmitter acetylcholine in the nerve cells and interfere with muscular responses and cause serious problems and sometimes fatal [ 35 ] .
analytical methods for determination of these toxic compounds are required to ensure that environment and human health will not be compromised by the usage of op pesticides .
there are many laboratory based analytical methods which are commonly used for the determination of op pesticides .
these include gas chromatography ( gc ) , high - performance liquid chromatography ( hplc ) , capillary electrophoresis [ 6 , 7 ] , mass spectrometry , and thermospray - mass spectrometry . besides the sensitivity and accuracy of these methods
, they are expensive and time - consuming and require pretreatment of the sample , as well as requiring highly trained persons to perform them , and are not suitable for in - field analysis [ 8 , 10 , 11 ] .
the need for rapid and cost - effective analytical methods for field analysis of op pesticides has developed many techniques , in which few of them are bioanalytical in nature .
enzyme - linked immunosorbent assay ( elisa ) is quite sensitive assay for op pesticides but , like other immunoassays , can detect only single compound and require multiple time - consuming steps .
but these methods are not well suited for process control monitoring assays where fast and repetitive analyses are required .
these problems can be solved by the use of biosensor technology which fulfils the demands for on - site monitoring and the rapid detection of neurotoxic agents .
biosensors based on the acetylcholine esterase ( ache ) inhibition have been widely used for the determination of op pesticides .
the main drawback of inhibition - based biosensors involves multiple - step operation , such as time - consuming incubation and reactivation / regeneration steps [ 14 , 17 ] .
organophosphorus hydrolase ( oph ) can catalyze the hydrolysis of a large number of op pesticides .
the enzymatic hydrolysis of op pesticides involves a ph change , as well as the generation of chromophoric species .
oph - based assays are direct and respond to op pesticides as substrate rather than inhibitors in case of inhibition - based biosensors .
the main problem associated with these methods involves the use of free enzyme which can be used once and also increases the cost of procedure . to overcome this problem , use of immobilization method for confining the enzyme molecules onto the different insoluble supports , this enhances the reusability of enzyme through immobilization .
although oph - based methods have several advantages such as selectivity for target analyte and show fast response , they are less sensitive and have low detection limits as compared to inhibition - based methods [ 20 , 21 ] .
these problems can be overcome by using the carbon nanotubes ( cnts ) as transducer material in amperometric biosensors .
deo et al . , which significantly enhanced the amperometric signals generated from op pesticides .
the oph biosensors provide a simple , fast , accurate , and selective monitoring of op pesticides , as well as being good for in - field applications and monitoring of detoxification processes . in the present method ,
the oph was extracted and purified from brevundimonas diminuta cells and used for the development of electrochemical biosensor by covalent immobilization of enzyme on to polyvinyl chloride ( pvc ) surface rather than working electrode .
this facilitates the opportunity to change the working electrode on deterioration , without wasting the immobilized enzyme which makes it economical and convenient .
the use of carbon nanotubes for the construction of working electrode provides efficient charge transfer from one phase to another .
the use of polyvinyl chloride ( pvc ) in bioanalytical techniques will open a new pathway in biosensor design and fabrication , diagnostic kits , and related biomedical instrumentation .
sephadex g-100 was purchased from mp biomedicals . nutrient broth , tris - hcl , edta , lysozyme , sucrose , and sodium hydroxide
glutaraldehyde ( grade 1.25% ) was purchased from sigma chemicals , and organophosphorus ( op ) pesticide ( methyl parathion ) was purchased from shri ram fertilizers & chemicals .
cyclic voltammetry ( cv ) measurements were performed on a potentiostat / galvanostat ( pgstat12/30/302 , autolab ) with a three - electrode system composed of a platinum ( pt ) wire as an auxiliary electrode , an ag / agcl electrode as reference electrode , and carbon nanotubes paste based working electrode .
uv spectrophotometer ( shimadzu corporation , japan ) , temperature controlled water bath ( sunron ) , digital ph meter ( eutech ) , refrigerator ( lg ) , microwave oven ( lg ) , rotatory incubator shaker ( hicon ) , magnetic stirrer ( hicon ) , refrigerated centrifuge ( sigma ) , and deep freezer ( voltas ) .
the study of surface morphology was carried out with scanning electron microscope ( sem ) ( model joel jsm-6510 , japan ) at department of chemistry , m. d. university , rohtak , india .
the lyophilized sample of bacteria brevundimonas diminuta ( mtcc 3361 ) was purchased from imtech , chandigarh .
lyophilized sample of b. diminuta was mixed with 5  ml of nutrient broth ( ph-8.0 ) in a test tube to make a bacterial suspension .
the conical flask ( 250  ml ) containing 100  ml nutrient broth ( ph-8.0 ) was inoculated with bacterial suspension .
the culture media were incubated in a rotatory incubator shaker at 200  rpm for 24 hours at 30c .
the cell pellets was incubated with 0.75  m sucrose , mixed in 7.5  ml of 50  mm sodium phosphate buffer , ph 8 at 20c for 10 minutes . the suspension war treated with lysozyme as described by herzliger et al .
the pallet was separated and re - suspended in 20  ml of 50  mm sodium phosphate buffer ( ph 8) containing 50  mm nacl at 4c for one hour with constant steering .
after centrifugation at 10000  rpm for 5 minutes the supernatant was separated and treated as crud enzyme solution .
purification of concentrated crude organophosphate hydrolase was done using sephadex g-100 column ( 2.5  30  cm ) as described by liu et .
the 0.1  ml of purified organophosphate hydrolase enzyme solution was mixed with 2.9  ml sodium phosphate buffer ( ph 8) , 5  nm methyl parathion ( 0.1  ml ) was added to the enzyme solution and incubated at room temperature for 10 minutes .
the total protein content of the purified organophosphate hydrolase enzyme solution was measured by lowery method .
( 1)op  pesticide+h2ooph4-nitophenol+diethyl  phosphate=410  nm 
  covalent immobilization of organophosphorus hydrolase onto pvc beaker surface was done by using the method described by hooda et al . .
inner surface of pvc beaker was filled with 5  ml of enzyme solution and kept overnight at 4c .
the reaction beaker was stored at 4c containing sodium phosphate buffer ( 50  mm , ph-8.0 ) . to confirm the immobilization of enzyme ,
the scanning electron microscopic ( sem ) study of the surface of pvc beaker was carried out , before and after enzyme immobilization at department of chemistry , m. d. university , rohtak , india .
carbon nanotubes powder ( 1.0  g ) and nh4cl ( 0.2  mg ) were mixed with paraffin oil in a ratio to obtain the consistency of paste .
this paste was filled in a plastic hollow tube ( 2  cm length and 4  mm diameter ) with one closed end .
electrical contact was made by inserting a silver wire into the carbon nanotubes paste at one end ( figure 1 ) .
the surface of the electrode was washed with buffer and stored at 4c when not in use .
an amperometric oph biosensor was developed by using enzyme immobilized pvc reaction beaker along with the three electrodes , that is , carbon nanotubes paste as working electrode , ag / agcl electrode as reference , and pt wire as auxiliary electrode and being connected through potentiostat / electrochemical analyzer ( figure 2 ) .
the cyclic voltammetric study of 4-nitrophenol was done in 50  mm sodium phosphate buffer ( ph-8.0 ) in a reaction beaker cell at room temperature . in the cyclic voltammetry experiments , the scan rate was 50  mv / sec and the scan range was 0  v to
+ 1.0  v. the following kinetic properties of oph biosensor were studied : optimum ph , incubation temperature , time of incubation , effect of substrate ( methyl parathion ) concentration , and calculation of k 
 m and i 
 max from line weaver - burk plot between reciprocal of substrate concentration ( 1/[s ] ) and reciprocal of amount of the current ( 1/[i ] ) .
the evaluation of present method was carried out by studying its linearity , minimum detection limit , analytical recovery , precision , and accuracy .
the linearity and minimum detection limit were calculated by correlating the values with standard graph . to determine the reliability of the method
, different concentrations of methyl parathion ( 5 and 10  m ) were added to the samples and the mean analytical recovery was determined by the present method . to study the reproducibility of the present method
, the op pesticide level was determined in the sample on the same day ( within batch ) and in the same sample after storage at 4c for one week ( between batch ) , coefficients of variation ( cvs ) were calculated for the present method . to evaluate the accuracy of present method , the levels of methyl parathion in 5 spiked water samples were determined by the present method and compared with those obtained by standard method . before reuse of the oph biosensor , it was washed by washing buffer ( 0.01  m phosphate buffer saline , ph-7.2 with 0.1% tween 20 ) .
the storage stability of the present biosensor was investigated over a period of two months , when reaction beaker was stored at 4c .
the amperometric response was also checked in the presence of potential interfering species such as glucose , fructose , and sucrose as well as metal ions ( zn ( ii ) , cu ( ii ) , cd ( ii ) , ni ( ii ) , and pb ( ii ) ) each at a concentration of 2  m .
the water and food samples prone to be contaminated with op pesticides determination were taken for study .
water samples which are used for the drinking purpose were used directly for the determination of op pesticides .
the food samples , that is , vegetables and fruits , ( cauliflower , cabbage , grapes , and apple ) were bought from local market .
100 grams of each chopped sample was crushed in pestle mortar and homogenize with 50  ml of pbs ( ph 7.0 ) and stirred for 1  h at room temperature .
the samples were filtered through filter paper and centrifuged at 8000  rpm for 8  min .
op compounds present in the sample were hydrolyzed to 4-nitrophenol by the oph enzyme immobilized onto pvc surface .
optimum potential was applied to oxidize 4-nitrophenol and the response current generated was directly proportional to the op compound concentration in the sample .
this electrochemical technique is employed to determine op pesticide concentration in food and water samples .
enzyme was purified from crude extract by gel filtration on sephadex g-100 and ion exchange chromatography on deae - sepharose .
the enzyme was purified by 14.15-fold to a specific activity of 20.81  u / mg of protein from the crude oph solution with a yield of 19.92% .
the purified enzyme showed a single band at 36  kda on sds - page .
covalent immobilization of enzyme on pvc beaker surface was carried out by method described previously .
inner surface of pvc beaker ( 30  ml ) capacity was treated with 2  ml of fuming nitric acid at 30c for about 2  h. after that pvc beaker was washed first with running tap water and then with double distilled water .
the surface was then treated with 1  ml of methyl cyanide for 1  h and thereafter with concentrated hcl ( 1  ml ) for 2  h. the activated surface of pvc was rinsed with distilled water and incubated with 1.25% ( w / v ) glutaraldehyde for about 8  h at 30c . the surface was then washed with distilled water to remove unbound glutaraldehyde .
finally , pvc beaker was poured with 5  ml of enzyme solution containing 0.245  mg of oph and kept overnight at 4c .
the morphology of pvc beaker surface before and after the immobilization of enzyme was studied by sem analysis .
the change on the chemically activated pvc surface ( figure 3 ) was observed after immobilization method .
such a change in surface morphology of the pvc surface after immobilization method confirms the enzyme immobilization .
the amperometric determination of op pesticides by oph - based biosensor depends on the anodic detection of 4-nitrophenol ( enzymatically liberated product ) by the electrode .
the working electrode showed a well - defined oxidation peak at a potential of + 0.8  v ( figure 4 ) .
therefore , + 0.8  v was selected as the working potential for the further studies .
the effect of ph on the biosensor response in 0.1  m succinate buffer ( ph 6.0 , 6.5 , and 7.0 ) , sodium phosphate buffer ( 7.5 , 8.0 , and 8.5 ) , and borate buffer ( ph 9.0 , 9.5 , and 10.0 ) in 6.010.0 range was studied .
the optimum ph for present method was 8.0 ( figure 5 ) which is higher than ph-7.4 [ 19 , 23 , 30 , 31 ] , ph 7.0 [ 32 , 33 ] , and lower than ph 8.8   and ph 9.5 .
the effect of the temperature on the biosensor response from 20c to 60c at a regular increase of 5c was also investigated .
the microenvironment provided by support used for immobilization makes it thermally stable and maintains its biological activity .
time of incubation was also studied from 5  min to 20  min at a regular increase of 5  min .
the response of the oph biosensor was increased up to 10  min after which no increase in response was observed .
the optimum incubation time for present method was 10  min ( figure 7 ) .
the effect of methyl parathion concentration on the response of present biosensor was studied up to 500  m at an interval of 50  m .
the present method showed a hyperbolic relationship between its response and methyl parathion concentration up to a final concentration of 400  m ( figure 8) after which no significant improvement in response was observed .
up to a concentration of 400  m , the oph biosensor showed a good response to methyl parathion . 

k 
 m and i 
 max were determined by calculating the slope and intercept for the reciprocal plot of current versus methyl parathion concentrations , that is , lineweaver - burk plot .
lineweaver - burk plot between the reciprocals of methyl parathion concentration and response current for biosensor was linear .
k 
 m and i 
 max were 322.58  m and 1.1  a ( figure 9 ) .
the linear working range and minimum detection limit of the present biosensor were calculated from standard graph between response current and substrate concentration ( methyl parathion ) . the linear range for methyl parathion
was found to be 0.1200  m . the reliability of the present method was determined by the analytical recovery of added methyl parathion ( table 1 ) .
the mean analytical recovery of added methyl parathion ( 5  m and 10  m ) was 98.6% and 99.1% , respectively .
to study reproducibility of the present method , the op pesticide level was determined in the sample repeatedly on the same day ( within batch ) and in the same sample after storage at 4c for one week ( between batch ) .
the results of within batch and between batch coefficients of variation ( cvs ) were < 1.58% and < 1.78% ( table 2 ) . to study the accuracy of the present method , the levels of methyl parathion in 5 spiked water samples were determined by the standard method ( x ) and by the present method ( y ) ( figure 10 ) .
the result obtained by the present method was in agreement with the standard method with a correlation of 0.985 .
the present biosensor lost 50% of its initial activity after its 25 uses over a period of 50 days ( figures 11 and 12 ) , when stored at 4c , which is better than previously reported biosensors [ 17 , 33 ] . among the various substances investigated for possible interference on the response of the present method , none caused any significant interference ( table 3 ) .
op pesticide level in fruits and vegetables was found in the range of 0.146  mg / kg0.243  mg / kg and 0.017  mg / kg0.031  mg / kg , respectively ( table 4 ) .
the op pesticide content of water samples was also determined and found in the range 0.00290.0091  mg / lit ( table 5 ) .
the use of carbon nanotubes as electrode material facilitates the enhanced amperometric determination of op pesticides .
the carbon nanotubes based transducer provides sensitive and stable detection of the 4-nitrophenol ( enzymatically liberated product of op pesticides ) .
the use of oph for biorecognition element and carbon nanotubes electrode for amperometric determination provides advantages over ache - based biosensors that are less specific towards op pesticides and requires multiple steps .
pvc has emerged as an ideal support for enzyme immobilization and its use in designing of present electrochemical cell has proved it better than other reported biosensors . the present method provides a laboratory based analytical method for the determination of op pesticides with high specificity .
the present method can act as a model for the development of indigenous op pesticide sensor in miniature form .
 polyvinyl chloride ( pvc ) can be used to develop reaction beaker which acts as electrochemical cell for the measurement of op pesticides . 
 being chemically inert , corrosion resistant , and easy in molding to various shapes and size , pvc can be used for the immobilization of enzyme . 
 organophosphorus hydrolase was immobilized covalently onto the chemically activated inner surface of pvc beaker by using glutaraldehyde as a coupling agent . 
 the carbon nanotubes paste working electrode was constructed for amperometric measurement at a potential of + 0.8  v. the biosensor showed optimum response at ph 8.0 with incubation temperature of 40c . 
 k 
 m and i 
 max for substrate ( methyl parathion ) were 322.58  m and 1.1  a , respectively . 
 evaluation study showed a correlation of 0.985 , which was in agreement with the standard method . 
 the oph biosensor lost 50% of its initial activity after its regular use for 25 times over a period of 50 days when stored in 0.1  m sodium phosphate buffer , ph 8.0 at 4c . 
 no interference was observed by interfering species . 
1. Introduction 
2. Materials and Methods
3. Results and Discussion
4. Conclusion