Skip to main content

Developing an in-house colorimetric method for detecting organophosphate and carbamate residue using cricket cholinesterase



Pesticides such as organophosphates and carbamates are used to control pests, leaving residues on fruits and vegetables. The assessment of residues in food materials is required for consumer and agricultural worker health protection. The purpose of this study was to develop an in-house colorimetric method for detecting organophosphate and carbamate residues using cricket cholinesterase that can be used as a low-cost, simple-to-use, and widely used test.


The method’s parameters, including substrate specificity, kinetics, and incubation time, were optimized. The results indicated that acetylthiocholine iodide was more active than butyrylthiocholine iodide. Using a buffer with a pH of 8.0 resulted in the greatest activity after 5 min. The same enzyme activity was observed at 25 °C and 30 °C, which was greater than at 35 °C and 40 °C. In this study, the Km and Vmax values for cricket cholinesterase were 3.75 mM and 35 µM/min, respectively. The specificity and purification factor of the produced ChE were 1.54 µM/min/mg and 1.43-fold, respectively. The limit of detection for detecting organophosphates and carbamates was in the range of 0.002–0.877 ppm. The validity of the method yielded the following results: relative sensitivity of 95%, relative specificity of 90%, relative accuracy of 93%, positive predictive value of 95%, and negative predictive value of 90%.


The developed method is simple and inexpensive, and it can analyze organophosphates and carbamates in a variety of vegetables at an appropriate detection level. Cholinesterase from cricket has the potential to be used for the development of a variety of methods for detecting residues, and the sensitivity could be enhanced using new techniques in the future.

Graphical Abstract


Pesticides such as organophosphates (OP) and carbamates (CM) are used to control pests. However, these chemicals can leave residues on crops after application, which can be harmful to human health if consumed in large quantities. They work by inhibiting acetylcholinesterase, an enzyme that is essential for the proper functioning of the nervous system. Chronic exposure to these pesticides has been linked to a range of health problems, including neurological and developmental disorders, cancer, and reproductive issues [1]. The assessment of residues in food materials is required for consumer and agricultural worker health protection.

Pesticide residues are currently identified using gas chromatography (GC) and high-performance liquid chromatography (HPLC). Due to the complexities of analysis and processing, these methods are either expensive or time-consuming. In most cases, sophisticated equipment is required, which is frequently unavailable in many analytical laboratories. Furthermore, these methods are unsuitable for large-scale data analysis [2,3,4,5]. Immunoassays are analytical methods that rely on an analyte interacting with an antibody that recognizes it with high affinity and specificity. However, this type of determination necessitates expensive equipment and highly skilled technicians. The majority of them used animals to produce the antibodies [6, 7]. Several enzyme-inhibition-based techniques and devices have been developed, including optical colorimetric assays, fluorometric assays, electrochemical biosensors, rapid test cards, and microfluidic devices [8,9,10,11,12]. Most of them were developed using the cholinesterase enzyme. A bioactive paper-based sensor was developed to detect acetylcholinesterase (AChE) inhibitors, such as organophosphate and carbamate pesticides, in food. Utilizing the Ellman colorimetric assay, this sensor incorporates a chitosan gel that is crosslinked with glutaraldehyde and includes both AChE and 5,5-dithiobis (2-nitrobenzoic) acid (DTNB). Acetylthiocholine iodide (ATCh) is used as an outside reagent. The assay is performed by immersing the paper strip in a pesticide solution and, after incubation, placing it in the ATCh solution. Enzyme catalysis of the substrate initiates a yellow color change, indicating the presence of AChE inhibitors. The sensor can detect certain pesticides with good detection limits and quick response times, demonstrating its potential as a rapid, sensitive, and cost-effective tool for pesticide detection [9]. In addition, a previous study explored the use of a thermostable esterase-2 (EST2) enzyme from Alicyclobacillus acidocaldarius as a biosensor for detecting organophosphate pesticides in food and the environment. Initial characterization of the enzyme involved determining its optimal pH, thermophilicity, and kinetics. The enzyme's activity was tested with several organophosphates, and it was found that paraoxon completely inhibits its activity. Notably, paraoxon and methyl-paraoxon significantly inhibited the fluorescence probe method. The enzyme biosensor was also successfully used to detect pesticides in actual food samples [13].

Acetylcholinesterase is an enzyme that rapidly hydrolyzes the neurotransmitter acetylcholine into choline and acetic acid at cholinergic synapses [14]. It plays an important role in pesticide residue detection as it is inhibited by organophosphate and carbamate pesticides [15]. While Butyrylcholinesterase (BChE) is an enzyme produced in the liver that degrades various compounds, including the neurotransmitter acetylcholine and various drugs. It is less effective than AChE at breaking down acetylcholine. Due to genetics and medical conditions, BChE levels can vary from person to person. In humans, plasma BChE decreases more rapidly than red blood cell AChE and recovers more rapidly following cessation of OP exposure [16].

Insect cholinesterase hydrolyzes acetylthiocholine slightly faster. The majority of the cholinesterase (ChE) activity in insects is found in the central nervous system compared to the peripheral nervous system. A special cholinesterase that hydrolyzes acetylthiocholine and is inhibited by physostigmine was discovered in the motor end plates of muscles in crickets. Insecticide application revealed that a critical site of action in the cricket's brain and nerve cord was involved in a knockdown [17]. Therefore, cholinesterase from cricket is an attractive alternative for developing methods to detect contamination with neurotransmitter-inhibiting pesticides, such as OP and CM.

Since the previous methods have some limitations for use as a screening test for pesticide exposure, such as high cost, high-tech equipment, and complexity, with in-house production of cholinesterase from cricket, which is widely available and inexpensive, combined with the development of a sample preparation method, the cost of the test can be reduced. Using common equipment such as a spectrophotometer in conjunction with an easy-step method can provide widespread use for screening residues, particularly in low-income countries.

The purpose of this study was to develop an in-house colorimetric method for detecting organophosphate and carbamate residues using cricket cholinesterase that can be used as a low-cost and simple-to-use test.

Materials and methods


Three different species of crickets were bought from a cricket farm in San Kamphaeng, Chiang Mai, Thailand, including Teleogryllus mitratus (Oriental ground cricket), Gryllus bimaculatus de Geer (African cricket), and Acheta domestica (House cricket). All crickets were frozen at − 20 °C prior to extraction.


Standard pesticides were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany), including dicrotophos, dichlorvos, mavinphos, carbaryl, carbosulfan, and methomyl. 5,5’-dithiobis-2-nitrobenzoic acid (DTNB), acetylthiocholine iodide (ATCh), and butyrylthiocholine iodide (BTCh) were purchased from Sigma-Aldrich.

Extraction of ChE from crickets and distribution of ChE content in different regions of the body

For comparing the activity of ChE in different regions of the body of three species of cricket, the extraction ratio at 1 mg sample: 8 mL of Phosphate Buffered Saline (PBS) buffer was prepared as 5 mg of the head, thorax, abdomen, and whole body of the cricket were each ground to a paste in a mortar and then resuspended in 40 mL of 1% Triton X-100 in PBS pH 7.2. Following that, 30 mL of the supernatant solution was collected after 30 min of performing a 10,000 g centrifugation at 4 °C. The extracted solution was maintained at − 20 °C for further measurement of ChE activity as described in “Measurement of ChE Activity”.

For protein precipitation, the extracted samples with the highest enzymatic activity were chosen. The protein was prepared using the salting out technique. To compress the solvation layer and increase protein–protein interactions, ammonium sulfate (NH4)2SO4 was used. Through aggregation and precipitation activity, increasing salt concentration caused the protein to separate and fall to the bottom of the sample solution [18]. To achieve the desired salt levels, 10 mL of the extract was mixed with an appropriate volume of saturated ammonium sulfate salt and then stirred at room temperature for 1 h using a magnetic stirrer. The centrifugation was carried out at 12,000 g for 30 min at 4 °C. After removing the supernatant, the protein pellet was resuspended in 2 mL of PBS, pH 7.2. The protein solution was kept at − 20 °C before being tested for protein concentration and ChE activity.

Preparation of the differently precipitated protein; The extracted sample was serially precipitated by preparing a 0–30% salt mixture before collecting the protein (Sample No. 1), as previously described. The supernatant was then prepared as a 30–50% salt mixture, and the second-step protein was collected (Sample No. 2). In the third step, the supernatant was collected and prepared to be a 50–75% salt mixture before collecting the protein (Sample No. 3). Samples No. 4, No. 5, and No. 6 were prepared to be 0–40%, 0–50%, and 0–75% salt mixtures, respectively. The protein was then collected in the manner described previously.

The Bradford’s Protein Assay [19] and the microwell plate technique were performed briefly, and protein standards ranging from 0.1 to 0.5 mg/mL were prepared in distilled water using a BSA standard. Ten µl of each protein standard was added to the wells of the 96 well plates, and 10 µl of distilled water (DW) served as a control. The samples were prepared in DW at three different dilutions of 1:10, 1:50, and 1:100 and added to the tested wells. The incubation time was set at 5 min at room temperature (25 °C) after preparing 1:5 Bradford reagent in DW and adding 300 µl of it to the tested wells, respectively. The absorbance at 595 nm was then measured, and the protein concentration of the samples was calculated using a standard curve obtained from five concentrations of standard BSA.

Measurement of ChE activity

The method was carried out using a modified version of the methods described in the Ellman assay, as well as some modifications based on our previous research. The Ellman method was used to detect this thiocholine using Ellman’s reagent, also known as DTNB. Thiocholine can break the disulfide bond in DTNB to produce 2-nitro-5-thiobenzoate ions, which have a yellow color and can be detected by their absorbance at 405 nm. As a result, the acetylcholinesterase enzyme activity can be measured by measuring the increase in yellow color over time [20, 21].

The overall reaction is

$${\text{Acetylthiocholine iodide }}\left( {{\text{C}}_{{7}} {\text{H}}_{{{16}}} {\text{INOS}}} \right)\, + \,{\text{H}}_{{2}} {\text{O}}\mathop{\longrightarrow}\limits^{{{\text{AChE}}}}{\text{Acetate }}\left( {{\text{CH}}_{3} {\text{COOH}}} \right)\, + \,{\text{Thiocholine }}({\text{C}}_{{5}} {\text{H}}_{{{14}}} {\text{INS}})$$
$${\text{Thiocholine }}\left( {{\text{C}}_{{5}} {\text{H}}_{{{14}}} {\text{INS}}} \right)\, + \,{\text{DTNB }}\left( {{\text{C}}_{{{14}}} {\text{H}}_{{8}} {\text{N}}_{{2}} {\text{O}}_{{8}} {\text{S}}_{{2}} } \right) \downarrow \to {2} - {\text{nitro}} - {5} - {\text{thiobenzoate }}\left( {{\text{C}}_{{7}} {\text{H}}_{{3}} {\text{NO}}_{{4}} {\text{S}}^{{ - {2}}} :{\text{ yellow product}}} \right)\, + \,{\text{other products}}$$

The cholinesterase activity was measured in the sample using 7.5 mmol/l ATCh as a substrate and 0.25 mmol/l DTNB as chromogen for measuring the ChE activity. Briefly, 10 µl of the sample was diluted in 240 µl PBS (pH 8.0) in a microplate, and then 70 µl of a mixture solution of 5:2 DTNB and substrate was added. The absorbance change at 405 nm was detected every 1 min for 5 min. The activity was calculated and reported in units per milliliter (units/mL), where one unit of ChE activity was defined as the amount of enzyme needed to hydrolyze 1 μmol of ATCh per min at 25 °C under the assay condition. Then, the calculations of specific activity (µM/min/mg), purification (fold), and yield (%) were performed [22].

Calculation of ChE activity

ChE activity (A) change/min as follows:

$$ChE \, activity \, \left( {units/mL} \right) \, = \, \Delta Absorbance \, x \, factor$$
$${\text{Factor }} = \, \left[ {{1}000/ \, \left( {{1}.{\text{36 x1}}0^{{4}} {\text{x 1}}} \right)} \right]{\text{ x dilution factor}}$$

where 1.36 × 104/M/cm = extinction coefficient of yellow anion; 1000 = conversion from mM/mL to /µM/mL. [20]

Optimization of the method for ChE activity test

As described in “Measuring ChE Activity,” 0–60 mM/mL substrates were used to determine the substrate type and rate of reaction for ATCh and BTCh substrates. The Michaelis–Menten graph was constructed. The Michaelis constant (Km) and the maximum velocity (Vmax) were read from the graph. The Michaelis–Menten model is one of the fundamental concepts in the field of enzymology, which is the study of enzymes. It describes the rate of enzymatic reactions by relating reaction rate (v) to substrate concentration ([S]).

The equation is represented as

$${\text{v }} = {\text{ Vmax}}\left[ {\text{S}} \right] \, / \, \left( {{\text{Km }} + \, \left[ {\text{S}} \right]} \right)$$

where v is the rate of the reaction. Vmax is the maximum rate achieved by the system, at maximum (saturating) substrate concentrations. [S] is the substrate concentration.

Km is the Michaelis constant, which is the substrate concentration at which the reaction rate is half of Vmax. It's an important measure of the enzyme’s affinity for the substrate; a smaller Km means a higher affinity, because less substrate is needed to achieve the maximum rate. The substrate that showed a lower Km and a higher Vmax was chosen for the next step [23].

The pH levels for the method were optimized. Six pH levels of PBS (pH 6.5–8.0) were used as the diluent, and ChE activity was measured using 7.5 mmol/mL ATCh as the substrate, as mentioned in the previous section. The results were compared, and the pH level of PBS with the highest activity was chosen for the next steps.

The temperature levels for the method were optimized. As mentioned in the previous section, ChE activity was measured in the water bath at 4 different temperatures (25–40 °C). The results were compared, and the temperature with the highest activity level was chosen for the following steps.

The effect of methyl alcohol on ChE activity was investigated. Six concentrations of methyl alcohol (MeOH) in PBS (0–5% v/v) were used as buffer solutions in ChE activity measurements. The results of ChE activity were compared, and the concentration of MeOH, which has no effect on ChE activity, was used in the step of pesticide extraction from vegetable samples.

Optimization of the method for ChE inhibition test

Acetylcholinesterase is a critical enzyme in the nervous system that degrades the neurotransmitter acetylcholine. Its function is critical to maintaining proper nerve cell communication. The chemical equation below represents the typical cholinesterase-catalyzed reaction:

$${\text{Acetylcholine }}\left( {{\text{C}}_{{7}} {\text{H}}_{{{16}}} {\text{NO}}_{{2}} \, + } \right)\mathop{\longrightarrow}\limits^{{{\text{AChE}}}}{\text{Acetic acid }}\left( {{\text{CH}}_{{3}} {\text{COOH}}} \right)\, + \,{\text{Choline }}({\text{C}}_{{5}} {\text{H}}_{{{14}}} {\text{NO}}\, + )$$

Organophosphates and carbamates, on the other hand, can bind to the active site of the cholinesterase enzyme, inhibiting its activity. When these pesticides bind to the active site of cholinesterase, they prevent it from breaking down acetylcholine. This results in an excess of acetylcholine, which causes overstimulation of certain bodily systems. A colorimetric assay can detect this change in cholinesterase activity. These assays typically use a substrate that changes color when cholinesterase acts on it. Under normal conditions, cholinesterase will catalyze the breakdown of this substrate, causing the solution’s color to change. This color change is reduced or absent when organophosphates or carbamates inhibit cholinesterase. It is possible to estimate the extent of cholinesterase inhibition and, thus, the presence of organophosphate or carbamate pesticides [24].

ChE activity was measured in the manner described in “Measurement of ChE Activity”. Four dilutions of extracted ChE (1:1–1:8) were used as the sample, and two concentrations of ATCh (7.50 and 3.75 mmol/mL) were used as the substrate. The absorbance of each reaction was measured. To further develop the ChE inhibition test, a combination of diluted ChE and concentrated ATCh with an absorbance difference of about 1 was chosen.

The optimal time for an inhibition test was performed briefly: 10 µl of cricket ChE was added into the well, followed by 100 µl of 1 part per million (ppm) mevinphos in 5% MeOH PBS pH 8.0 as an inhibitor, and 140 µl of PBS pH 8.0, and then incubated at 6 different times (10–40 min). The activity of ChE was measured, as mentioned in the previous section, at room temperature (25 °C) and pH 8.0 conditions. The inhibition percentages were calculated, and the appropriate incubation time was used in the method for ChE inhibition by OP and CM pesticides. The inhibition percentage was calculated by

$$\% \, Inhibition = \frac{E0 - E}{{E0}} \times 100$$

where [E0] is the initial enzyme activity in the absence of an inhibitor, and [E] is the enzyme activity in the presence of an inhibitor.

ChE inhibition by organophosphate and carbamate pesticides

Ten µl of cricket ChE was added into the well, followed by 100 µl of the inhibitors OPs and CMs at different concentrations (0.005–5.000 ppm) in 5% MeOH PBS pH 8.0 and 140 µl of PBS pH 8.0, and incubated for 30 min. The activity of ChE was measured, as mentioned in the previous section, at room temperature (25 °C) and pH 8.0 conditions. The calibration curve was obtained, and the half maximal inhibitory concentration (IC50) and 25% inhibition concentration (IC25) were calculated using the Prism program version 4.01. The IC25 was used as the limit of detection (LOD) of this method.

Extraction of pesticides from vegetable samples

Five grams of each vegetable sample were chopped finely and shaken for 5 min with 10 mL of dichloromethane. Five milliliters of the extract were transferred to a glass tube and dried in hot water to completely remove the solvent. The pellet was resuspended with 200 µl of dichloromethane, then 200 µl of 5% MeOH PBS, pH 8.0, was added and vigorously mixed. Dichloromethane was then completely removed using hot water. The extracted solution was used in a further step of ChE activity measurement.

ChE inhibition by organophosphate and carbamate pesticides in vegetables

The pesticide-free vegetables (cabbage), confirmed to have no OP residues by gas-chromatography coupled with flam photodetector (GC–FPD) as described in the previous study [25], were spiked with pesticides at several concentrations (0.005–5.000 ppm) of dicrotophos, dichlorvos, mevinphos, carbarly, carbosolfan, and methomyl. All samples were extracted as mentioned in the previous section, and the developed method was performed to determine an inhibition. Briefly, 10 µl of the twice diluted ChE and 100 µl of the sample were added into a microplate, then 140 µl PBS (pH 8.0) was added and incubated at room temperature (25 °C) for 30 min. In a further step, 70 µl of a mixture solution of 5:2 DTNB and 3.75 mmol/mL ATCh substrate was added. The absorbance changes at 405 nm were detected every 1 min for 5 min at 405 nm. The inhibition was calculated as described previously. The calibration curve was obtained, and the IC50 and IC25 were calculated using the Prism program version 4.01. The IC25 was used as the LOD of this method.

Application of developed method to vegetable samples

The 30 vegetable samples examined using GC–FPD came from our previous study [26], such as kale (Brassica alboglabra L.H. Bailey), cabbage (Brassica oleracea var. capitata L.), bok choy (Brassica rapa. L.), long bean (Vigna unguiculata ssp. Sesquipedalis), and morning glory (Ipomoea aquatica Forssk.). All samples were extracted, as mentioned in the previous section, to determine their inhibition using the developed method. The inhibition was calculated in the manner mentioned previously. Samples showing an inhibition test result greater than 25% were interpreted as having the presence of an insecticide. For relative sensitivity, relative specificity, relative accuracy, positive predictive value, and negative predictive value, the results are calculated and compared with GC–FPD results [27].

Statistical analysis

ChE activity results were reported as the mean and standard deviation. The inhibition curve was conducted, and the IC50 was calculated using GraphPad Prism 4.01. A one-way ANOVA was used to compare ChE activity and inhibition percentage across the study. The statistical tests were two-sided, with a significance level of 0.05. The SPSS statistical program, version 17, was used for the analyses.


House cricket cholinesterase activities

According to the results in Table 1, ChE activity in the house cricket was found to be superior to others in every body region, with the whole body of cricket showing the best at 11.97 ± 0.53 units/mL. In this study, the whole body of the house cricket was used for enzyme production.

Table 1 ChE activity in different body regions in three cricket species

Table 2 displays the protein concentration and enzyme activity of PBS-extracted samples and precipitated proteins. PBS-extracted total protein had the highest concentration (95.50 mg), whereas 0–50% precipitated protein exhibited the highest enzyme activity (27.75 units/mL), followed by 0–40% precipitated protein (27.70 units/mL). Specific activity and purification were calculated, and the 0–40% salt-precipitated protein had the highest specific activity (1.54 µM/min/mg) and purification factor (1.43-fold). The yield percentage of the 0–50% salt-precipitated protein yield was the highest at 53.91%, followed by the 0–40% and 0–75% salt-precipitated protein yield percentages at 53.81% and 53.07%, respectively. According to the highest specific activity and purification factor, the 0–40% salt-precipitated protein of the house cricket was used to determine substrate specificity and kinetics in subsequent steps.

Table 2 Summary of protein concentration and ChE activity results from crude purification at different salt levels

Substrate specificity and kinetics

The activity of ChE on ATCh and BTCh substrates was determined using the selected sample (0–40% salt-precipitated protein). The result from the Michaelis–Menten graph showed the Vmax and Km on ATCh substrate at 35 units/mL and 3.75 mM/mL, respectively. While the Vmax and Km on BTCh substrate showed 1.80 units/mL and 3.75 mM/mL, respectively, as shown in Fig. 1, the outcome indicated that the ChE of house cricket is more specific to ATCh than BTCh substrate.

Fig. 1
figure 1

Michaelis–Menten graph: the result of ChE activities on (A) ATCh substrates and (B) BTCh substrates

According to Fig. 2, pH conditions of 6.5 showed low activity of ChE, whereas 7.0, 7.5, and 8.0 showed high activity of ChE. In addition, the ChE activity showed no significant difference in pH 7.0–8.0. The pH of 8.0 was chosen for further steps according to its highest activity. The comparison of temperature levels was performed, and the result showed the same activity of enzymes at 25 °C and 30 °C, which was higher than at 35 °C and 40 °C, as shown in Fig. 3. According to the convenience of the method that can be performed without the incubator, a temperature of 25 °C was used for the method in the next step.

Fig. 2
figure 2

Optimization results of the method: (A) pH levels and (B) temperature levels

Optimization of the method for ChE inhibition

The effects of several concentrations of MeOH (0–5% V/V) in phosphate buffer solution pH 8.0 were tested. The result showed no difference in ChE activity between the concentrations of 0–5% V/V MeOH, as shown in Table 3. That means we can use 5% MeOH in PBS pH 8.0 as the resuspension solution for the extracted samples.

Table 3 Result of ChE activity in the 6 levels (%V/V) of MeOH in PBS pH 8.0

To select the appropriate combinations of dilution of the produced ChE and substrate concentration for the inhibition method, the optimized ChE activity test method was performed in a variety of combinations. The combination of 1:2 diluted ChE and 3.75 mM/mL ATCh exhibited an absorbance difference of approximately 1.0 at 5 min, as shown in Table 4. This combination was used in the subsequent step of the inhibition test procedure. To optimize the inhibition method, the time required for pesticides and ChE to react completely was determined. At 30 min, the inhibition of ChE by 1 ppm mevinphos was complete, and there was no significant difference, thereafter, as shown in Table 5.

Table 4 Results of the absorbance difference of the combinations of ChE dilution and ATCh concentration
Table 5 Result of inhibition percentage of 1 ppm Mevinphos pesticide in difference incubation time

Development and application of ChE inhibition method for vegetable samples

After utilizing the optimized ChE inhibition method, the IC50 and IC25 of OP and CM pesticides were determined. In this study, the IC25 was used as the LOD for the inhibition method, and the LOD values for standard OPs and fortified samples ranged between 0.004–0.246 ppm and 0.009–0.426 ppm, respectively. LODs for standard CMs and fortified samples range between 0.001–0.497 ppm and 0.002–0.508 ppm, respectively. As shown in Table 6, mevinphos was the most potent inhibitor among OP pesticides, while carbosulfan was the most potent inhibitor among CM pesticides.

Table 6 Detection limits of the standard pesticides and fortified samples using the ChE inhibition method

According to Table 7, the true positive, true negative, false positive, and false negative values for the evaluation of the validity of pesticide detection methods using the developed ChE inhibition method versus the GC–FPD method in 30 vegetable samples were 19, 9, 1, and 1, respectively. Consequently, the relative sensitivity was 95%, the relative specificity was 90%, the relative accuracy was 93%, the positive predictive value was 95%, and the negative predictive value was 90%.

Table 7 Efficiency of the developed ChE inhibition method for the detection of insecticides in vegetable samples compared with GC–FPD


Several studies have demonstrated that ChE can be extracted from a variety of animals and insects, and their substrate specificity and kinetics have been investigated. Most of them were used in the development of pesticide detection methods. Several purification techniques, including ammonium sulfate precipitation, Sephadex chromatography, DEAE–cellulose chromatography, gel filtration, affinity chromatography, procainamide affinity chromatography, etc., were combined to prepare ChE for use in their studies [22, 28,29,30,31,32,33,34]. In these previous studies, the specificity and purification fold ranged from 9.0–1336.7 µM/min/mg to 4.8–3897 fold, respectively. Higher specificity and purification are associated with a more complex and expensive technique. Since the objective of this study was to develop a low-cost in-house method, one step of crude purification (ammonium sulfate precipitation) was used to extract ChE from crickets. The specificity and purification of the produced ChE were 1.08 µM/min/mg, and 1.54-fold, respectively. The lower specificity of this study is due to the ineffectiveness of the ammonium sulfate precipitation method in isolating different protein sizes compared to the other previously mentioned methods. Nevertheless, one step of crude purification can be used as an easy and low-cost technique for the ChE production method in this study.

The optimal pH and temperature of 8.0 and 25 °C were confirmed. Previous studies that reported identical findings for insect cholinesterase supported the findings [17, 28]. This study revealed that, under optimal conditions, the LOD of standard pesticides was lower than that of pesticide-spiked samples for both OPs and CMs, which may be caused by the vegetable matrix (i.e., color and acidity) and the extraction process (i.e., solvent). Since this study demonstrated that the LOD for OPs and CMs were between 0.002 and 0.508 ppm, it is comparable to the LOD of a paper-based acetylcholinesterase inhibition assay using commercial acetylcholinesterase for the detection of OPs, which was between 0.003 and 0.600 ppm [35]. The results were superior to the qualitative acetylcholinesterase detection kit based on a modification of the Ellman and ELISA methods, using honeybee (Apis mellifera L.) heads as the source of ChE with a LOD range of 0.50–4.80 ppm [36]. Furthermore, the newly developed smartphone-based colorimetric sensor technique demonstrated that the low LOD of a method for all pesticides was less than 1.5 × 10(-7) M, indicating greater sensitivity than the U.S. Environmental Protection Agency regulations specify [37]. Indicating that the cricket ChE can be used in method development and is a potential source for the future.

A method of ChE inhibition for monitoring OP and CM pesticides in vegetable extracts was presented. Importantly, the ChE inhibition method was validated in vegetable matrices, and its performance was confirmed by an accredited GC–FPD. The short analysis time (20 min for sample preparation for 10 samples and 30 min for the ChE inhibition method) and low cost of the 96-microwell plate demonstrated that the ChE inhibition method can be used by laboratories with limited resources as a preliminary screening tool. In addition, 48 samples (2 wells/sample in a 96-microwell plate) were able to be analyzed using the developed high-throughput method. In the test, samples of vegetables with an intense dark color showed a false positive, which the extraction method was unable to clear. Indicating that color is one of the matrices in this method, along with other chemicals that can influence ChE, such as heavy metals [38]. Low levels of pesticide residues in the samples led to false negative results. Since it yielded greater than 90% of all efficiency indicators, especially 95% of the positive predictive value, our developed method can be used as a screening test for detecting OP and CM residues in vegetable samples.

Multifaceted approaches can improve cholinesterase (ChE) assay sensitivity and recovery. Concentrating target pesticides and eliminating interfering substances requires improving sample preparation methods. Optimizing assay conditions such as temperature, pH, and incubation time increases enzyme activity. Enzyme enhancers may benefit, while nanotechnology-based methods may improve sensitivity and stability. Microfluidics or lab-on-a-chip technologies may enable efficient, miniaturized testing. Finally, advanced instrumentation such as spectrometry or chromatography could improve detection.


The results of this study demonstrate conclusively that the developed method can analyze OP and CM pesticides at appropriate detection levels in many types of vegetables. The method requires minimal equipment and is simple enough for use in a primary laboratory equipped with a spectrophotometer. The developed method can be easily integrated into pesticide control programs as a screening method. Future method development could utilize cricket as a source of ChE.

Availability of data and materials

All data generated and analyzed during this study are included in this manuscript.







Gas chromatography


High-performance liquid chromatography


Gas chromatography coupled with flam photodetector






5,5’-Dithiobis-2-nitrobenzoic acid


Acetylthiocholine iodide


Butyrylthiocholine iodide


Bovine serum albumin

IC50 :

The half maximal inhibitory concentration

IC25 :

The 25% inhibition concentration


The limit of detection


Part per million


  1. De Silva HJ, Samarawickrema NA, Wickremasinghe AR. Toxicity due to organophosphorus compounds: what about chronic exposure? Trans R Soc Trop Med Hyg. 2006;100(9):803–6.

    Article  PubMed  Google Scholar 

  2. Pinheiro Ade S, de Andrade JB. Development, validation and application of a SDME/GC-FID methodology for the multiresidue determination of organophosphate and pyrethroid pesticides in water. Talanta. 2009;79(5):1354–9.

    Article  PubMed  Google Scholar 

  3. Hongsibsong S, Prapamontol T, Xu T, Hammock BD, Wang H, Chen ZJ, et al. Monitoring of the organophosphate pesticide chlorpyrifos in vegetable samples from local markets in northern Thailand by developed immunoassay. Int J Environ Res Public Health. 2020.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Asensio-Ramos M, Hernandez-Borges J, Gonzalez-Hernandez G, Rodriguez-Delgado MA. Hollow-fiber liquid-phase microextraction for the determination of pesticides and metabolites in soils and water samples using HPLC and fluorescence detection. Electrophoresis. 2012;33(14):2184–91.

    Article  CAS  PubMed  Google Scholar 

  5. Ulusoy HI, Koseoglu K, Kabir A, Ulusoy S, Locatelli M. Fabric phase sorptive extraction followed by HPLC-PDA detection for the monitoring of pirimicarb and fenitrothion pesticide residues. Mikrochim Acta. 2020;187(6):337.

    Article  CAS  PubMed  Google Scholar 

  6. Jin RY, Guo YR, Wang CM, Wu JX, Zhu GN. Development of a bispecific monoclonal antibody to pesticide carbofuran and triazophos using hybrid hybridomas. J Food Sci. 2009;74(1):T1-6.

    Article  CAS  PubMed  Google Scholar 

  7. Razak CN, Salam F, Ampon K, Basri M, Salleh AB. Development of an ELISA for detection of parathion, carbofuran, and 2,4-dichlorophenoxyacetic acid in water, soil, vegetables, and fruits. Ann N Y Acad Sci. 1998;864:479–84.

    Article  CAS  PubMed  Google Scholar 

  8. Cao J, Wang M, Yu H, She Y, Cao Z, Ye J, et al. An overview on the mechanisms and applications of enzyme inhibition-based methods for determination of organophosphate and carbamate pesticides. J Agric Food Chem. 2020;68(28):7298–315.

    Article  CAS  PubMed  Google Scholar 

  9. Badawy ME, El-Aswad AF. Bioactive paper sensor based on the acetylcholinesterase for the rapid detection of organophosphate and carbamate pesticides. Int J Anal Chem. 2014;2014:536823.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Cao J, Wang M, She Y, Abd El-Aty AM, Hacimuftuoglu A, Wang J, et al. Rapid colorimetric determination of the pesticides carbofuran and dichlorvos by exploiting their inhibitory effect on the aggregation of peroxidase-mimicking platinum nanoparticles. Mikrochim Acta. 2019;186(6):390.

    Article  PubMed  Google Scholar 

  11. Kaewmaroeng T, Wunnapuk K, Chockjamsai M, Sringarm K, Hongsibsong S. Simple Colorimetric method for cholinesterase-inhibitor screening in gastric content by using phytoesterase enzyme from kidney bean. Pak J Biol Sci. 2020;23(7):904–10.

    Article  CAS  PubMed  Google Scholar 

  12. Qian S, Lin H. Colorimetric sensor array for detection and identification of organophosphorus and carbamate pesticides. Anal Chem. 2015;87(10):5395–400.

    Article  CAS  PubMed  Google Scholar 

  13. Garefalaki V, Manco G, Porzio E. Use of biosensors for rapid and sensitive detection of pesticides in food samples for food safety chemical risk assessment. EFSA J. 2022;20(Suppl 2):e200922.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Trang A, Khandhar PB. Physiology, acetylcholinesterase. Treasure Island: StatPearls; 2022.

    Google Scholar 

  15. Lionetto MG, Caricato R, Calisi A, Giordano ME, Schettino T. Acetylcholinesterase as a biomarker in environmental and occupational medicine: new insights and future perspectives. Biomed Res Int. 2013;2013:321213.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Nigg HN, Knaak JB. Blood cholinesterases as human biomarkers of organophosphorus pesticide exposure. Rev Environ Contam Toxicol. 2000;163:29–111.

    CAS  PubMed  Google Scholar 

  17. Booth GM, Lee AH. Distribution of cholinesterases in insects. Bull World Health Organ. 1971;44(1–3):91–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Duong-Ly KC, Gabelli SB. Salting out of proteins using ammonium sulfate precipitation. Methods Enzymol. 2014;541:85–94.

    Article  CAS  PubMed  Google Scholar 

  19. Hammond JB, Kruger NJ. The bradford method for protein quantitation. Methods Mol Biol. 1988;3:25–32.

    CAS  PubMed  Google Scholar 

  20. Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88–95.

    Article  CAS  PubMed  Google Scholar 

  21. Sawarng N, Hongsibsong S, Sapbamrer R, Wongta A, Tongjai P. Effectiveness of a participatory program on pesticide use behavior and blood cholinesterase levels in Chiang Mai Province, Northern Thailand. J Environ Public Health. 2021;2021:6746367.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Ma E-B, He Y-P, Zhu KY. Comparative studies of acetylcholinesterases purified from two field populations of the oriental migratory locust (Locusta migratoria manilensis): implications of insecticide resistance. Pestic Biochem Physiol. 2004;78(1):67–77.

    Article  CAS  Google Scholar 

  23. Rodriguez J-MG, Hux NP, Philips SJ, Towns MH. Michaelis-Menten graphs, lineweaver-burk plots, and reaction schemes: investigating introductory biochemistry students’ conceptions of representations in enzyme kinetics. J Chem Educat. 2019;96(9):1833–45.

    Article  CAS  Google Scholar 

  24. Colovic MB, Krstic DZ, Lazarevic-Pasti TD, Bondzic AM, Vasic VM. Acetylcholinesterase inhibitors: pharmacology and toxicology. Curr Neuropharmacol. 2013;11(3):315–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Amekawa Y, Hongsibsong S, Sawarng N, Yadoung S, Gebre GG. Producers’ perceptions of public good agricultural practices standard and their pesticide use: the case of Q-GAP for cabbage farming in Chiang Mai province, Thailand. Sustainability. 2021.

    Article  Google Scholar 

  26. Wongta A, Sawang N, Tongjai P, Jatiket M, Hongsibsong S. The assessment of organophosphate pesticide exposure among school children in four regions of Thailand: analysis of dialkyl phosphate metabolites in students’ urine and organophosphate pesticide residues in vegetables for school lunch. Toxics. 2022.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Monaghan TF, Rahman SN, Agudelo CW, Wein AJ, Lazar JM, Everaert K, et al. Foundational statistical principles in medical research: sensitivity, specificity, positive predictive value, and negative predictive value. Medicina. 2021.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Lee A-H, Metcalf RL, Kearns CW. Purification and some properties of house cricket (Acheta domesticus) acetylcholinesterase. Insect Biochem. 1974;4(3):267–79.

    Article  CAS  Google Scholar 

  29. Rao M, Dasgupta D. Purification of the enzyme acetylcholinesterase (AChE. 3.1. 1.7) from Meloidogyne incognita and Heterodera Zeae. Revue de nématologie. 1991;14(4):517–24.

    CAS  Google Scholar 

  30. Gao J-R, Zhu KY. An acetylcholinesterase purified from the greenbug (Schizaphis graminum) with some unique enzymological and pharmacological characteristics. Insect Biochem Mol Biol. 2001;31(11):1095–104.

    Article  CAS  PubMed  Google Scholar 

  31. Tham L, Perumal N, Syed M, Shamaan N, Shukor M. Assessment of clarias batrachus as a source of acetylcholinesterase (AChE) for the detection of insecticides. J Environ Biol. 2009;30(1):135–8.

    CAS  PubMed  Google Scholar 

  32. Ding Y, Wu X, Fang J. Purification and characterization of acetylcholinesterase from brain tissues of oreochromis aurea and its application in environmental pesticide monitoring. Sci Cold Arid Reg. 2011;3(4):339–43.

    Google Scholar 

  33. Ma M, Zhang B, Li S-C. Purification and partial characterization of acetlcholinesterase from pardosa astrigera L. Koch. J Cell Animal Biol. 2011;5(1):11–6.

    Google Scholar 

  34. Yang Y-x, Niu L-z, Li S-n. Purification and studies on characteristics of cholinesterases from daphnia magna. J Zhejiang Univ Sci B. 2013;14(4):325–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Apilux A, Isarankura-Na-Ayudhya C, Tantimongcolwat T, Prachayasittikul V. Paper-based acetylcholinesterase inhibition assay combining a wet system for organophosphate and carbamate pesticides detection. Excli J. 2015;14:307–19.

    PubMed  PubMed Central  Google Scholar 

  36. Kim BM, Hwang TE, Jin LT, Kim YS, Shim JH. Development of an acetylcholinesterase-based detection kit for the determination of organophosphorus and carbamate pesticide residues in agricultural samples. Bull Korean Chem Soc. 2007;28:929–35.

    Article  CAS  Google Scholar 

  37. Zhao T, Liang X, Guo X, Yang X, Guo J, Zhou X, et al. Smartphone-based colorimetric sensor array using gold nanoparticles for rapid distinguishment of multiple pesticides in real samples. Food Chem. 2023;404(Pt B):134768.

    Article  CAS  PubMed  Google Scholar 

  38. Frasco MF, Fournier D, Carvalho F, Guilhermino L. Do metals inhibit acetylcholinesterase (AChE)? Implementation of assay conditions for the use of AChE activity as a biomarker of metal toxicity. Biomarkers. 2005;10(5):360–75.

    Article  CAS  PubMed  Google Scholar 

Download references


We gratefully acknowledge support from the Research Institute for Health Science, Chiang Mai University, 50200, Thailand, Multidisciplinary Research Institute, Chiang Mai University, 50200, Thailand, and Faculty of Science, Asia–Pacific International University, Saraburi 18180, Thailand.


Not applicable.

Author information

Authors and Affiliations



Conceptualization, AW and SH; methodology, AW and PA; validation, AW, PA and UJ; formal analysis, AW and NS; resources, AW and SH; data curation, AW and PS; writing—original draft preparation, AW; writing—review and editing, AW, SH, NS, WW and PD; visualization, AW, SH, WW and PD; supervision, SH, WW and PD; All authors read and approved the final manuscript.

Corresponding author

Correspondence to Anurak Wongta.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have approved to submit this work to Chemical and Biological Technologies in Agriculture. They declare that there is no conflict of interest in relation to the submission of the article.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wongta, A., Hongsibsong, S., Anand, P. et al. Developing an in-house colorimetric method for detecting organophosphate and carbamate residue using cricket cholinesterase. Chem. Biol. Technol. Agric. 10, 65 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: