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Development of an organophosphate and carbamate pesticide test kit by using a magnetic particle coated with esterase enzyme from honey bee heads


Organophosphates (OPs) and carbamates (CMs) play a vital role in Thai agriculture, public health, and pest control. However, their detection presents challenges due to expensive methods requiring specialized expertise. Addressing this gap, our study introduces an innovative, cost-effective method for detecting OPs and CMs in Thailand. We utilized the test kit by using magnetic particles coated with esterase enzymes from honey bee heads combined with a colorimetric approach. The developed test kit shows low limits of detection (LODs) at 0.001 mg/L of dichlorvos (OPs) and 0.004 mg/L for carbaryl (CMs). The efficiency of the developed test kits in comparison with the standard technique of gas chromatography with flame photometry detection (GC-FPD) shows a comparable result in fruit and vegetable residue analysis. This developed test kit proposes a cost-effective OPs and CMs analysis test kit for Thailand, promising expense reduction and simplified verification processes. Its applicability to fruits and vegetables signifies practicality in agriculture, marking a significant advancement in accessible and efficient pesticide residue monitoring. Despite challenges in pesticide use, our method holds promise for improving safety and efficiency in Thai pesticide management.

Graphical Abstract


Thailand's agricultural operations have undergone significant transformations marked by increased reliance on heavy machinery, artificial chemicals, hybrid seeds, and technology. These changes, aimed at boosting crop yields and pest control, have, however, led to new challenges. The increased use of pesticides, including organophosphates (OPs) and carbamates (CMs), poses serious environmental and health concerns [1]. Thailand's reliance on pesticides has increased significantly, with annual import increases covering a wide range of pesticides, such as chlorpyrifos (OPs) and carbaryl methomyl (CMs) [2]. The extensive use of pesticides results in the release of harmful residues, posing a significant risk to both the environment and public health [3]. OPs and CMs can enter the human body through various routes, including the skin, eyes, gastrointestinal system, respiratory system, and wounds. Once inside the body, these chemicals can distribute across tissues and organs through the bloodstream, leading to detrimental effects [4]. OPs have been linked to cancer, neurological issues, liver problems, and other health issues, while CMs are associated with reproductive toxicity, respiratory conditions, carcinogenicity, and neurological problems [5, 6].

Agricultural pollution of OPs and CMs has direct implications for food safety, contributing to an increase in food safety incidents globally [7]. Recognizing this, the World Health Organization (WHO) considers food safety a crucial public health concern [8]. Traditional analytical methods such as gas chromatography (GC), high-performance liquid chromatography (HPLC), and mass spectrometry (MS) are employed to ensure food quality and safety [9,10,11,12]. However, in recent years, biological detection systems, particularly biosensors based on esterase enzyme inhibition, have gained prominence for their simplicity and cost-effectiveness [13].

Honey bees serve as excellent indicators of environmental pesticide contamination due to their foraging behaviour and sensitivity to chemical pollutants [14]. The esterase enzyme from honey bees has been identified as a valuable biological indicator for evaluating the impact of OPs and CMs [15]. The decrease in esterase enzyme activity indicates the presence of inhibitors, making it an effective tool for pesticide detection [16]. In this context, magnetic nanoparticles (MNPs) have emerged as promising precursors for biosensor development. MNPs, with their large surface-to-volume ratio, enable nanoscale reactions, increased sensitivity, and easy recoverability using external magnetic fields [17, 18]. Unlike traditional techniques, MNPs do not require extensive sample pre-treatment, ensuring high operational speed and accuracy [19].

The basics of detecting organophosphate and carbamate with extracted enzymes

The enzyme extracted from honey bee heads serves as a pivotal catalyst in the conversion of alpha-naphthyl acetate (ANA) to α-naphthol. The ensuing α-naphthol rapidly engages with fast blue B salt, resulting in the formation of a distinctive purple diazonium dye [20], as illustrated in Fig. 1. The quantification of enzymatic activity is established through the elucidation of a linear relationship between the absorbance of the purple diazonium dye and time. Notably, the concentrations of organophosphates and carbamates are deduced by assessing the inactivation percentage of the enzyme, as delineated by Eq. 1. This method presents an effective approach for the quantification of OPs and CMs, coupling enzymatic activity with a discernible colorimetric response for precise and reliable detection.

Fig. 1
figure 1

The colorimetric reaction of esterase enzyme with naphthyl acetate and fast blue B Salt

Herein, the present research focuses on the development and optimization of a test kit for detecting OPs and CMs, involving the fixation of esterase enzyme on magnetic microbeads. The innovative approach of covalent coupling on magnetic microbeads followed by colorimetric detection enhances accuracy, offering a rapid and cost-effective solution for detecting pesticide residues in agricultural products [20, 21]. The study evaluates the specificity, practicality, and stability of the developed colorimetric sensors, highlighting their potential as valuable tools for enhancing food safety.

Materials and methods

Chemicals and stock solutions

The chemicals and reagents employed in this study, including Alpha naphthyl acetate (ANA), fast blue B salt, and sodium dodecyl sulfate (SDS), were procured from reputable sources such as Sigma-Aldrich (USA), Chemipan (Thailand), and RCI Labscan (Thailand). Notably, 95% ethanol was obtained from Chemipan, sodium chloride, disodium hydrogen phosphate anhydrous, monosodium phosphate, and dimethyl sulfoxide from RCI Labscan, and potassium chloride from Supelco (Germany). The homogenization process was conducted using an IKA homogenizer (model T 25 digital), and the assays were carried out in 96-well plates (Corning, USA), with measurements performed on a Tecan microplate reader (Switzerland). Organophosphorus (OPs) and carbamate (CMs) insecticides were acquired from Dr. Ehrenstorfer (Augsburg, Germany). Pesticide stock solutions, prepared at a concentration of 10 − 0.0001 mg/L in 5% methanol in PBS at pH 7.2, were stored at 4 °C. Additional chemicals, including ferric chloride (FeCl3), ferrous chloride (FeCl2), ammonium chloride (NH4Cl), N-hydroxysuccinimide (NHS), and 3,3'-dichloro-1,1'-biphenyl-4-ylidenemaleimide (EDC), utilized for the activation of magnetic particles, were sourced from RCI Labscan (Thailand) and Sigma-Aldrich.

Sample gathering

Dwarf honey bee (Apis florea F. and A. andreniformis) specimens were meticulously gathered from a village situated at coordinates 18° 53′ 32.0" N and 98° 49′ 56.9" E. The collection site, located approximately one kilometer from these geographical coordinates, served as a representative habitat for the studied bee population. To preserve the integrity of the collected specimens, they were promptly stored in a freezer at -80 °C prior to the extraction process.

Esterase extract from honey bee heads

The extraction of the esterase enzyme followed the methods of Chen et al. [21]. In brief, 1 g of honey bee heads is followed by homogenization with 30 mL of PBS at pH 7.2 using a T-25 digital Ultra-TURRAX® homogenizer. Subsequently, the homogenized sample underwent a 16-h incubation at 4 °C and was later centrifuged at 10,000 rpm at 4 °C for 20 min.

Synthesis of magnetic nanoparticles (MNPs) by the co-precipitation method

The synthesis of magnetic nanoparticles followed the methods outlined by RĂCUCIU et al. [22]. In brief, 289 mg of FeCl3·6H2O (1.10 mmol) and 114 mg of FeCl2·4H2O (0.57 mmol) were dissolved in 20 mL of distilled water. The solution was then sonicated for 10 min and poured into a four-neck flask equipped for stirring the reaction under a nitrogen atmosphere with constant agitation. Control the temperature at 60 °C, stir the solution for 30 min, then add 6 mL of conc. NH4OH (28% w/w) by slowly dropping it into the solution, and continue stirring for 2 h as shown in the mechanism (1). Then inculcate the solution to cool to room temperature, vacuum the black particles from the solution with a magnetic stick, and wash the particles with water that is distilled until it is neutral. The synthesized particles were collected as a suspension by adding distilled water and sonicating for 30 min. The concentration of the suspended particles was determined (mg/mL) by aspirating 0.5 mL of the particles into a glass vial. It was dried at a temperature of 55 °C for 24 h and then weighed to calculate the weight of particles in a volume of 1 ml (mg/mL).

$${\text{2FeCl}}_{{3}} + {\text{FeCl}}_{{2}} + {\text{ 4H}}_{{2}} {\text{O }} + {\text{ 8NH}}_{{3}} \to {\text{ Fe}}_{{3}} {\text{O}}_{{4}} + {\text{ 8NH}}_{{4}} {\text{Cl}}$$

Immobilization of magnetic nanoparticles by covalent coupling

The immobilization of magnetic nanoparticles, as outlined by Meng et al. [23], involves a meticulous process aimed at enhancing the stability and functionality of the resulting enzyme-bound nanoparticles. Initially, 114.6 mg of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 69 mg of N-hydroxysuccinimide (NHS) are dissolved in 10 mL of a solution containing magnetic nanoparticles (MNPs) at a concentration of 10 mg/mL. This solution, referred to as the activated solution, undergoes gentle mixing at 4 °C overnight. EDC and NHS play pivotal roles in this process: EDC acts as a zero-length crosslinker, facilitating the activation of carboxyl groups on the surface of the MNPs, while NHS enhances the reactivity of these activated groups by forming NHS esters. The resultant activated solution provides an ideal substrate for the subsequent coupling reaction. Following activation, the enzyme extract obtained from honey bee heads is added to the MNPs in a 1:4 ratio and stirred at 4 °C overnight. The enzyme extract contains biologically active enzymes that are essential for catalyzing specific biochemical reactions. By coupling these enzymes onto the surface of the MNPs, their stability and longevity are greatly enhanced, allowing for prolonged and efficient catalytic activity. After the coupling reaction, the solution is carefully removed, and the enzyme-bound MNPs are thoroughly washed with cold phosphate-buffered saline (PBS) at pH 7.2. This washing step helps remove any unbound or loosely bound enzyme molecules, ensuring the purity and integrity of the immobilized enzyme-NP complexes. Finally, the remaining enzyme-bound MNPs are suspended in 10 mL of PBS at pH 7.2 and stored at 4 °C until further use.

Measuring enzyme activity

Esterase enzyme activity was assessed following the modified spectrophotometer method as outlined by Yang et al. [24]. First, 30 μL of MNP-Enzyme solution was introduced into the Eppendorf tube, subsequently isolating only the magnetic nanoparticles (MNPs). Following this, 150 μL of a solution comprising 5% methanol in PBS at pH 7.2 was thoroughly mixed. Subsequently, 50 μL of alpha-naphthyl acetate (ANA) was introduced into the reaction mixture, and after a 15-min incubation at room temperature, 25 mL of a 1% fast blue B salt solution was added to a 5% sodium dodecyl sulfate (SDS) solution. The dynamic alteration of the blue color resulting from the enzymatic reaction was quantified at 595 nm using a microplate reader, providing a comprehensive analysis of esterase enzyme activity.

Experimental design

For the optimization of the reaction, a 17-set Box Behnken design employing three independent parameters with three levels each was selected using Stat Ease Design Expert Software. The chosen independent variables, namely the percentage of methanol (%MeOH), temperature (°C), and pH, are recognized as pivotal factors influencing the enzymatic activity in the reaction. These variables were systematically investigated at three distinct levels: low (coded as − 1), medium (coded as 0), and high (coded as + 1), as delineated in Table 1. This experimental design allows for a comprehensive exploration of the parameter space, enabling the identification of optimal conditions for enhancing the desired enzyme activity in the reaction.

Table 1 Levels and the experimental condition for Box Behnken design

Inhibition test

The method's sensitivity in detecting organophosphates (OPs) and carbamates (CMs) was following the protocol from Jaitham et al. [20]. To evaluate the sensitivity of the method in detecting organophosphates (OPs) and carbamates (CMs), 150 μL of pesticides, ranging in concentrations from 10 to 0.0001 mg/L (ten-fold dilution), were introduced into Eppendorf tubes containing magnetic particles. The mixture was incubated at room temperature (25 °C) for 15 min. Subsequently, 50 μL of 4 mM alpha-naphthyl acetate (ANA) in ethanol was added, followed by another round of mixing and incubation at room temperature (25–30 °C) for an additional 15 min. The subsequent addition of 25 μL of 1% fast blue B salt in a 5% sodium dodecyl sulfate (SDS) solution induced a color change in the tube. The resultant color alteration was quantified at a wavelength of 595 nm using a microplate reader. The percentage of enzyme inhibition was computed, representing the 50% inhibitory concentration (IC50) in accordance with Eq. 2. The calculated IC50 values were graphically depicted using a prismatic graph plate program, offering a comprehensive visualization of the sensitivity of the method across varying pesticide concentrations.

$${\text{I }} = \, \left( {{\text{E}}0 \, {-}{\text{ E1 }}/{\text{ E}}0} \right) \, \times { 1}00$$

where: [I] represents the inhibition percentage of enzyme activity, [E0] denotes the enzyme activity of the control group, and [E1] signifies the enzyme activity of the test group.

Development of an enzyme-based detection method

Serving as a biomarker, the discernible change in color within the tested solution serves as an indicator of the presence of organophosphate (OPs) and carbamate (CMs) groups. In practical application, MNPs-Enzyme was blended with standard carbaryl pesticide at varying concentrations (10, 0.1, 0.01, 0.001, and 0.0001 mg/L). This mixture underwent a 15-min incubation period at room temperature, followed by further blending with 4 mM alpha-naphthyl acetate in ethanol and an additional 15-min incubation. Subsequently, 1% fast blue B dye in a 5% sodium dodecyl sulfate (SDS) reagent was introduced. The assessment of test results involved a visual comparison of the color observed in the sample test tube against that of the blank tube. This qualitative color change offers a straightforward and effective means of identifying the presence and concentration of OPs and CMs, providing a rapid and accessible diagnostic approach.

Fourier transform infrared spectroscopy – attenuated total reflectance (FTIR-ATR)

In this study, FTIR-ATR spectra were obtained from three samples: MNPs, MNPs-enzyme, and MNPs-after inhibition. All samples were lyophilized, frozen, and then freeze-dried using the SP Scientific freeze-dry system (at − 55 °C, pressure of 0.02 mbar for 96 h). To homogenize the sample, it was ground into a fine powder using an agate mortar. The spectra were measured using a Perkin-Elmer FTIR spectrometer equipped with a platinum ATR that holds a diamond ATR module. The spectra were recorded in reflective mode from 4000 to 400 cm−1 at a resolution of 4 cm−1, averaging 24 scans for each measurement. The sample was carefully removed from the optics using a dust brush, followed by cleaning the optics with solvent and acetone before applying the next sample.

Pesticide residue extraction from vegetable samples

Extraction from vegetable samples followed the protocol outlined by Jaitham et al. [20]. In brief, vegetable samples were meticulously collected, minced, and subsequently homogenized to ensure uniformity. Subsequently, 10 g of the homogenized samples were carefully transferred to a 50 mL centrifuge tube, followed by the addition of 10 mL of acetonitrile. The sample was manually shaken for 5 min to facilitate thorough extraction. Following this, the pipet solution was transferred to another tube and allowed to evaporate until it reached complete dryness. The dried residue was then reconstituted by adding 5% methanol in PBS pH 7.2 to the tube, ensuring the dissolution of any pesticides present. Extracted samples obtained from the test tubes were then subjected to analysis using the previously described procedures.

Organophosphate pesticide analysis in vegetable samples

  • Sample Removal and Cleaning: Vegetable samples weighing 5 g were put into a 50 mL centrifuge tube using an extraction technique derived from Sapbamrer and Hongsibsong (2014) [25]. In brief, 5 μg/mL triphenylphosphine internal standard (IS) and 10 mL of high-performance liquid chromatography-grade acetonitrile were then added. Following a 5-min centrifugation at 2500 rpm, the supernatant was transferred to a fresh tube holding 3 g of NaCl and 6 g of MgSO4. After another centrifugation cycle, the extract was dried at 30 to 35 °C in a vacuum rotary evaporator. Five milliliters of ethyl acetate were used to reconstitute the dried residue. The ethyl acetate phase was pipetted into two dispersive solid-phase extraction tubes in a 1 mL aliquot, which was then centrifuged. The resulting extract was evaporated with nitrogen, reconstituted in 1.0 mL of 10% methanol in PBS at pH 7.0 for immunoassay, and another portion was reconstituted with 0.5 mL of ethyl acetate for gas chromatography (GC) analysis.

  • Gas Chromatography-Flame Photometric Detection (GC-FPD): For GC analysis, a Hewlett-Packard model 6890 with a flame photometric detector and a capillary column (DB-1701, 0.25 mm, 30 m length, 0.25 μm film thickness, Agilent J & W column, Agilent Technologies, USA) were utilized. The injection port's temperature was adjusted to 220 °C in spitless mode. The oven's temperature was programmed to start at 100 °C for 10 min, ramp up to 180 °C at a rate of 15 °C per minute for 5 min, reach 250 °C at a rate of 5 °C per minute for 3 min, and end at 290 °C for 4 min. The carrier gas consisted of 99.99% helium.


Magnetic nanoparticles (MNPs) synthesis by co-precipitation method

This study presents the synthesis of magnetic nanoparticles (MNPs) through the co-precipitation method involving ferrous ions (Fe2+) and ferric ions (Fe3+) under basic conditions (pH 9–10). Equation 1 indicates that ammonium hydroxide was used as the precipitating agent. As seen in Fig. 2, the resultant MNPs were a fine brown-black powder with unique magnetic characteristics that made it possible for a magnetic rod to readily absorb them. This unequivocally confirms the magnetic characteristics of the synthesized iron nanoparticles. The use of ammonium hydroxide in the co-precipitation process highlights its effectiveness as a coagulant. It assists in the effective synthesis of MNPs with appropriate magnetic properties.

Fig. 2
figure 2

Magnetic nanoparticles

Structure analysis of MNPs using FTIR-ATR technique

In the analysis of three types of magnetic nanoparticles, including MNPs, MNPs-enzyme, and MNPs-after inhibition, the FT-IR technique was utilized, and distinctive signals were observed on the particles, as depicted in Fig. 3. Specifically, signals in the range of 500–700 cm1 were identified as metal–oxygen bond (Fe–O) stretching vibrations, characteristic of iron oxide nanoparticles. For iron (II) chloride (FeCl2), typical FTIR-ATR bands associated with Cl-Cl stretching and Fe-Cl bonding were found around 1625 cm1 for the Cl-Cl stretching band. Similarly, in the case of iron (III) chloride (FeCl3), the Cl-Cl stretching band also appeared around 1625 cm1 due to the presence of three chloride ions per formula unit. Ammonium chloride (NH4Cl) exhibited an N–H bond stretching around 3500 cm1.

Fig. 3
figure 3

FT-IR Spectrum of MNPs Coated with Esterase Enzyme from Honey Bee Heads

For MNPs-Enzyme and MNPs-After inhibition, signals emerged at 1700 cm−1, indicative of enzyme activity cleaving ester bonds. This position aligns with the expected stretching vibrations of ester linkages in the enzyme molecule. Concurrently, bands related to metal oxides (Fe–O) persisted around 500–700 cm−1 for iron oxide nanoparticles. Further, the incorporation of EDC (3,3'-dichloro-1,1'-biphenyl-4-ylidene maleimide) in the synthesis process introduced characteristic bands related to the carbonyl group (C = O) at approximately 1750 cm−1, and NHS (N-hydroxysuccinimide) contributed bands related to the hydroxyl group (H–O) around 3400 cm−1. When combined, these findings provide a comprehensive understanding of the structural components and interactions found in artificial magnetic nanoparticles. It provides insight into the effects of inhibition on enzyme coating, observed vibrational spectra, and the successful integration of metal oxides.

Efficiency of enzymes from honey bee heads in response to organophosphate and carbamate

This investigation delved into enzymatic activity extracted from honey bee heads of pesticide-sensitive bees, focusing on two local bee species, Apis florea F. and A. andreniformis. It is well known that these two species of bees are susceptible to commonly used insecticides, organophosphate, and carbamate in the region. The study revealed distinct variations in enzymatic properties exhibiting significant inhibition, indicating heightened sensitivity to dichlorvos pesticide. The calculated IC50 value for Apis florea F. was measured at 0.02 mg/L, in stark contrast to A. andreniformis, which displayed a higher IC50 value exceeding 0.1 mg/L. This marked difference underscores a clear variation in susceptibility to organophosphate and carbamate pesticides between the two bee species. The significance of these findings lies in emphasizing the crucial need to consider species-specific factors when utilizing honey bee enzymes for pesticide detection applications.

Application of response surface methods to optimize enzymatic activity in the reaction

This study makes use of Response Surface Methods, more especially, Design-Expert 13, to maximize the amount of enzyme activity in a process. The three main parameters are A (the percentage of methanol, %MeOH, at 2%, 5%, and 8%), B (the reaction temperature at 20 °C, 30 °C, and 40 °C), and C (the pH at 5, 7, and 9). These are all shown in Table 1. The study emphasizes how important enzymatic activity is by using these different factors; the three-dimensional response surface is shown. A graphic depiction of the methodical analysis of enzymatic activity with respect to pH, %MeOH, and temperature can be seen in Fig. 3.

Interestingly, changes in reaction pH correspond to a noticeable increase in Abs (nm). However, the most favorable outcomes are attained at pH 7, precisely within a reaction environment comprising 5% MeOH and temperatures ranging between 25 and 30 °C will show enzyme activity of 0.74 units/mL.

Inhibition of extracted esterase by pesticides (OPs and CMs)

Following Eq. 2, the calculation of enzyme activity inhibition percentages occurred at different concentrations of carbamates (CMs) and organophosphates (OPs). Through the utilization of a prismatic graph plate application, values were systematically derived. Table 2. presents the IC50 values obtained. Figure 4. visually displays OP concentrations, while Fig. 5. illustrates the IC50 values of CMs. These diagrams provide a comprehensive depiction of the correlations between pesticide response and enzyme activity, along with the inhibitory effects resulting from varying doses of OPs and CMs. They also highlight the effectiveness of these substances in impacting enzymatic activities (Fig. 6).

Table 2 IC50 values of organophosphate and carbamate as inhibitors of magnetic-coated enzymes from honey bee heads
Fig. 4
figure 4

Systematic Evaluation of Enzymatic Activity in the Reaction

Fig. 5
figure 5

The susceptibility to OPs is determined by the inhibition values of the 50% enzyme (IC50). a The IC50 graph for dichlorvos is 0.02 mg/L. b It shows the colors produced by inhibition from dichlorvos

Fig. 6
figure 6

The susceptibility to CMs is determined by the inhibition values of the 50% enzyme (IC50). a The IC50 graph for carbaryl is 0.02 mg/L. b It shows the colors produced by inhibition from carbaryl


FT-IR analysis of magnetic nanoparticle synthesis, enzyme coating, and inhibition

In comparing MNPs-enzyme with MNPs-after inhibition using FT-IR analysis, clear distinctions emerged, notably in the absence of specific signals within MNPs and MNPs-after inhibition. In MNPs synthesized without incorporating the enzyme, NHS, or EDC, the FT-IR spectra displayed a pronounced absence of signals linked to enzyme-related activities, particularly the characteristic 1700 cm−1 band indicative of ester bond cleavage by enzyme activity. The expected signals associated with EDC and NHS, including the carbonyl group (C = O) at approximately 1750 cm−1 for EDC and the hydroxyl group (H–O) around 3400 cm−1 for NHS, were notably absent. Similarly, in MNPs-After inhibition, where enzyme activity was successfully suppressed, the FT-IR spectra also lacked signals associated with enzyme-related activities. The absence of the 1700 cm−1 band suggests the effective impact of the inhibition process on the enzyme ability to cleave ester bonds. Furthermore, the characteristic bands related to EDC and NHS were not evident, supporting the notion that the inhibition process affected the expected chemical interactions. This absence of signals related to enzyme activity, NHS, and EDC in both MNPs, and MNPs-After inhibition underscores the specificity and success of the synthesis and inhibition processes. It highlights the controlled manipulation of magnetic nanoparticles' surface chemistry, emphasizing the impact of each step in the synthesis and inhibition protocols. These findings offer valuable insights into the tailored design and functionality of magnetic nanoparticles, particularly in applications involving enzymatic activities and inhibitory processes.

Response surface methods (RSM) and design-expert 13 for optimizing enzymatic activity within a reaction

Enzymatic activity is maximized by using Design-Expert 13 and other response surface methods. Through investigating the connection between pH, MeOH percentage, and reaction temperature. For a comprehensive reference, the three-dimensional reaction surface depicted in Fig. 3. shows the optimal conditions for the activity of enzymes. The enzyme exceptional sensitivity to pH fluctuations is shown by the significant rise in Abs (nm) with changes in reaction pH. At pH 7, 5% methanol, and 25–30 °C, the highest enzymatic activity was observed. This implies a precisely calibrated set of parameters that enhance the effectiveness of the enzyme process. Notably, the study shows that pH variations have a major impact on the enzyme. The result emphasizes how vital it is to maintain pH equilibrium for optimal results. Additionally, the reaction conducted at room temperature is beneficial for the development of a practical test kit. The fact that %MeOH has little effect on the response improves the situation even further, constructing an enzyme system that is immune to variations in this parameter. All in all, these findings provide a solid foundation for developing a trustworthy test kit, particularly considering the normal environmental conditions seen in Thailand.

Inhibition of extracted esterase by pesticides (OPs and CMs)

The analysis of organophosphates (OPs) and carbamates (CMs) using magnetic particle-coated enzyme esterase extracted from honey bee heads has shown significant improvements in the limit of detection (LOD). For example, the LOD for OP Dichlorvos was determined to be 0.001 mg/L, while for CMs, the LOD for carbaryl was found to be 0.004 mg/L. Compared to prior studies presented in Table 3, these results demonstrate a substantial enhancement in the LOD for Dichlorvos. Previous investigations detected Dichlorvos using esterase extract from crickets [26, 27], which showed detectability LOD at 0.110 and 0.246 mg/L, respectively, and honey bee heads [20], which detected it at 0.003 mg/L. In contrast, the developed method utilizing magnetic bead-coated enzyme esterase extracted from honey bee heads achieved a significantly lower LOD of 0.001 mg/L. This marked improvement in sensitivity is noteworthy, particularly when considering maximum residue limits for pesticides (MRL).

Table 3 Comparison between the limit of detection (LOD) achieved by the honey bee esterase extraction and the magnetic-coated enzyme from honey bee heads

Furthermore, in comparison with the LOD of 0.003 mg/L reported in previous studies by Jaitham et al. in 2023 [20], where enzyme extract from honey bee heads was utilized, it becomes apparent that although the enzyme extract could detect Dichlorvos at a low level, the magnetic particle-coated enzyme esterase extraction method underscores the superior sensitivity conferred by the magnetic particle-coated enzyme esterase extraction method. These findings strongly suggest that the magnetic particle contributes to a heightened level of sensitivity, particularly when utilized in conjunction with enzyme esterase extracted from honey bee heads. This advancement holds promise for more accurate and reliable detection of pesticides, assuredly contributing to enhanced environmental monitoring and pesticide residue management.

Testing real samples for residues using a test kit with a magnetic particle-coated esterase enzyme from honey bee heads

To investigate the possibility of creating an organophosphate and carbamate pesticide test kit, a thorough examination of pesticide residues in various vegetable samples purchased from the neighborhood market was conducted. We examined 31 gathered vegetable samples using a dual technique that combined gas chromatography with flame photometric detection (GC-FPD) and magnetic particle coating with esterase enzymes from honey bee heads. Table 4. presents an overall favorable picture based on the results, revealing that most of the examined vegetables had undetectable pesticide levels. Nevertheless, the use of GC-FPD revealed the existence of residues of ethidium bromide and feroxides in quantities ranging from 0.03 to 0.27 mg/kg in the vegetable samples.

Table 4 The efficiency of the developed magnetic-coated enzyme from honey bee heads for the detection of insecticides in vegetable samples compared with GC-FPD

Subsequent evaluation using the developed magnetic particle coated with esterase enzyme from honey bee heads demonstrated a high level of performance with a sensitivity of 90%, a specificity of 95%, an accuracy of 93%, a positive predictive value of 90%, and a negative predictive value of 95%. Despite the overall commendable performance of the test kit, it did reveal a specific limitation in identifying certain pesticides. A comparative analysis between GC-FPD and the test kit results brought to light significant disparities in pesticide residue detection across the 31 vegetable samples.

GC-FPD identified pesticide residues in 32.26% of the samples, leaving 67.74% with no detectable residues. In contrast, the test kit displayed a marginally lower positive detection rate of 29.03% but notably excelled in negative detection, reaching 70.97%. These findings underscore the potential of the developed test kit for specific pesticide residue detection. However, the observed limitation signals the necessity for further exploration and refinement of the test kit's capabilities. While demonstrating effectiveness in certain scenarios, a nuanced consideration of the test kit's specificity and sensitivity is crucial for a comprehensive pesticide residue analysis of diverse agricultural products.


In addition to the aforementioned achievements, this study's novel outcomes include the successful demonstration of the magnetic particle-coated enzyme esterase extraction method's superior sensitivity in detecting organophosphates (OPs) and carbamates (CMs), as exemplified by the remarkably low limit of detection (LOD) of 0.001 mg/L for the OP Dichlorvos. This heightened sensitivity, attributed to the synergistic use of magnetic beads and enzyme esterase extracted from honey bee heads, represents a significant advancement over conventional techniques, promising enhanced accuracy and reliability in pesticide detection. However, the present research encountered several challenges, notably in optimising synthesis and inhibition protocols to ensure reproducibility and reliability across diverse environmental conditions. Addressing these challenges will be crucial for further refining the methodologies developed in this study and enhancing their practical applicability. Looking toward future directions, there are several promising avenues for research. Firstly, efforts should focus on expanding the scope of analytes targeted by the magnetic particle-coated enzyme esterase extraction method to encompass a broader range of pesticides and environmental pollutants. Additionally, ongoing refinement of protocols and methodologies will be essential to improving sensitivity, specificity, and robustness, thereby facilitating broader real-world applications in environmental and agricultural contexts. Moreover, exploring potential synergies between magnetic particles and other innovative technologies could yield further improvements in pesticide detection methodologies. Collaboration with interdisciplinary fields such as materials science, nanotechnology, and biochemistry may uncover novel approaches for enhancing sensitivity and selectivity while also addressing challenges related to sample preparation and analysis. Ultimately, by overcoming present challenges and pursuing future research directions, this study's findings hold great promise for advancing environmental monitoring, pesticide residue management, and ensuring food safety in agricultural practices.


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Authors would like to extend our thanks to our research team and Nuchareeya Tata, a student intern, for their invaluable help and support throughout the analysis process.


This work was partially supported by the Research Institute for Health Sciences, Chiang Mai University, grant number 010/2566 and TGCMU2566P002/2566.

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UJ, BC, KD, and SH designed the experiment and wrote the manuscript. UJ, PT, PJ, PY and SY performed the experiment. BC, and SH supervised the study and provide funding. All authors contributed to the article and approved the submitted version.

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Correspondence to Bajaree Chuttong or Surat Hongsibsong.

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Jaitham, U., Kawichai, S., Yadoung, S. et al. Development of an organophosphate and carbamate pesticide test kit by using a magnetic particle coated with esterase enzyme from honey bee heads. Chem. Biol. Technol. Agric. 11, 77 (2024).

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