Universal solvent-adaptive fluorescent AZ NPs-ACh sensor for reliable visual detection of organophosphorus pesticides via AChE-H 2 O 2 signaling

1 Introduction

Organophosphorus pesticides (OPs), a class of highly effective and broad-spectrum pesticides, have played a crucial role in global agricultural production since the 1940s, safeguarding the world’s food and herbal production (Costa, 2018). However, their inherent high toxicity poses a persistent and serious threat to human health and the ecological environment. The primary toxicological mechanism of OPs involves the irreversible phosphorylation of serine residues (Ser) at the active site of acetylcholinesterase (AChE), a crucial enzyme in the nervous system (Sirin and Zhang, 2014). This modification completely inhibits AChE’s capacity to recognize and hydrolyze its substrate, acetylcholine (ACh), resulting in permanent enzyme inactivation (Robb et al., 2025). AChE is a key enzyme in the cholinergic nervous system that terminates nerve impulse transmission by efficiently hydrolyzing the neurotransmitter ACh, and its activity is essential for maintaining nervous system homeostasis (Leuzinger, 1969; McHardy et al., 2017). Inactivation of AChE leads to excessive accumulation of ACh in the synaptic cleft, causing persistent overstimulation of cholinergic receptors and a clinical condition known as cholinergic crisis. Symptoms affect the respiratory, cardiovascular, and central nervous systems and may progress to respiratory failure or death in severe cases (Hulse et al., 2014; Figueiredo et al., 2018; Aroniadou-Anderjaska et al., 2020). It is estimated that globally about 3 million people are poisoned by OPs each year, resulting in nearly 300,000 deaths (Faria et al., 2015). It is estimated that approximately 3 million people worldwide are poisoned by OPs each year, resulting in nearly 300,000 deaths (Faria et al., 2015). In addition to acute toxicity, long-term or low-dose exposure to OPs has been linked to various chronic health problems, including neurodegenerative diseases, psychiatric disorders, and potential carcinogenic risks (Adeyinka et al., 2025). Therefore, a visual detection method suitable for complex matrices of OPs residues in food, natural products and the environment has crucial scientific significance and social value for protecting public health and maintaining food safety.

To address the serious challenges posed by OPs residues, establishing reliable analytical methods to ensure compliance with national maximum residue limits (MRLs) has become a research focus in the field of analytical chemistry (Boyapati, 2020). Techniques such as gas chromatography-tandem mass spectrometry (GC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely recognized as the gold standard methods for multi-residue pesticide analysis due to their high sensitivity, selectivity, and accuracy (Hernández-Mesa and Moreno-González, 2022). Notwithstanding these advantages, they present several limitations: sample preparation is often laborious and time-consuming, requires large volumes of organic solvents, and depends on specialized laboratory instrumentation, which limits their applicability for on-site analysis and complex sample matrices (Tripathy et al., 2017). Furthermore, the analysis of complex materials such as traditional Chinese medicines (TCM) is often accompanied by co-extracted endogenous compounds (e.g., pigments, volatile oils, saponins, and alkaloids), leading to significant matrix effects (Dang et al., 2024; Liu, 2024; Zhang et al., 2024). These effects interfere with ionization efficiency during mass spectrometry analysis, thereby reducing the accuracy and reliability of quantitative analysis (Zhang et al., 2022; Ji et al., 2023). Fluorescence-based assays have emerged as a promising alternative for enzyme activity monitoring and inhibitor screening, benefiting from simple, rapid analysis, minimal solvent usage, and real-time detection capabilities (Inês and Cosme, 2025). Substantial advances have been made in the development of fluorescent probes for AChE activity sensing, most of which rely on enzyme-specific catalytic reactions to trigger fluorescence turn-on responses (Chao et al., 2016; He et al., 2021; Fortibui et al., 2022). However, these approaches also face notable constraints: they are prone to interference from solvent effects, especially in samples pre-treated using QuEChERS methodologies. Elevated concentrations of organic solvents, such as acetonitrile or methanol, significantly increase background fluorescence, reducing detection accuracy (Lee et al., 2024). Furthermore, the complex synthesis methods of many sensors, coupled with intricate detection procedures, hinder on-site implementation of testing (Li Q. et al., 2025; Ryu et al., 2025; Wang et al., 2025). Additionally, many existing probe designs do not emit within optical windows suitable for visual on-site inspection, thereby limiting their utility in practical scenarios. Consequently, to overcome two key challenges inherent in current methods—severe solvent interference and limited field applicability—the development of a field-capable solvent-resistant fluorescence detection platform is a valuable direction of scientific and practical significance.

In this work, we propose an innovative dual-advantage sensing strategy (Scheme 1). A reaction system comprising AChE and ChOx was designed to linearly convert AChE activity into stable, detectable H₂O₂ signals. Building on this, a novel fluorescent sensor (AZ NPs-ACh-sensor) responsive to H₂O₂ was synthesized. This sensor not only exhibits a highly specific fluorescence change response to H₂O₂, but also maintains excellent fluorescence stability in various high-concentration organic solvents, demonstrating robust solvent resistance. Compared to recent pesticide residue detection sensors, this sensor also offers the advantage of simpler synthesis (Jahandideh-Roudsari et al., 2025; Li H. et al., 2025; Song et al., 2025). Furthermore, after fixing the sensor onto a paper substrate, its large Stokes shift and common excitation wavelength (365 nm) enable convenient on-site detection using a portable ultraviolet lamp. This approach offers greater convenience compared to detection methods requiring specialized workstations (Siripongpreda et al., 2025; Wei et al., 2026). Therefore, this dual-advantage design offers a novel solution with broad application prospects for the rapid and accurate analysis of OPs residues in complex matrices such as TCM, enabling visual on-site detection.

2 Experimental section

2.1 Reagents and instruments

2.1.1 Reagents

Zn(NO₃)₂·6H₂O, HAuCl₄, AgNO₃, bovine serum albumin (BSA), NaOH, PBS buffer and H₂O₂ were purchased from Sigma-Aldrich. Acetylcholinechloride (ACh) was purchased from MCE. AChE, ChOx were purchased from Macklin Biochemical Technology Co., Ltd., Shanghai. Triazophos purchased from Tianjin Alta Technology Co. Chenpi samples were provided by Shenzhen Institute of Drug Control. Hydrogen Peroxide Content Detection Kit by Shanghai Yuanye company.

2.1.2 Instruments

Fluorescent detection was performed using a microplate reader (Thermo Scientific Varioskan Flash, Switzerland). UV vis absorbance was measured using a UV-2450 UV-vis spectrophotometer (Shimadzu Company, Japan). Infrared data is sourced from the NICOLET IS5 infrared detector (Thermo Scientific, United States) and XPS data was obtained from the DIONEX AQUION XPS detector (Thermo Scientific, United States).Besides, TEM images were collected by transmission electron microscopy (FEI Talos F200X G2, United States). DLS and zeta characterization were performed on a Zeta sizer Nano-Z instrument (Malvern, United Kingdom). Synthesis of sensor by BDA-1002 ultrasonic cleaner Ltd. and temperature and speed double digit magnetic (heating plate) stirrer (ZNCL-BS, China). Intuitive fluorescence changes captured by UV lamp 254/366 nm light box (CAMAG, 022.9160, Switzerland).

2.2 Preparation of AZ NPs-ACh-sensor

Configure 50 mg/mL BSA solution 10 mL, add 4 mL, 10 mM HAuCl₄ and 0.5 mL, 20 mM AgNO₃ solution, stir vigorously for 10 min. After adding 1 mL, 1 M NaOH solution was immediately transferred to a thermostatic stirrer and stirred vigorously at 37 °C for 12 h. Collected AuAg NCs and dialyzed for 48 h. The post-dialysis product was placed in a clean glass container and stored at 4 °C away from light.

AZ NPs -ACh-sensor was prepared by taking 4 mL of the product and adding 10 mL 30 mM Zn(NO₃)₂·6H₂O to sonicate for 30 min. The product was collected by centrifugation, washed twice with methanol and once with ultrapure water, dry with nitrogen gas, and set aside for use.

2.3 Detection of H₂O₂ by AZ NPs-ACh-sensor

The sensor concentration of 30 mg/mL was configured to react with 10 mM H₂O₂ solution, which was added to a 96-well plate into the enzyme marker (ex = 310 nm/em = 690 nm) at different times to optimize the optimal reaction time. Afterwards, the reaction was carried out with different concentrations of H₂O₂ solution, which was added to a 96-well plate and then measured by an enzyme marker (ex = 310 nm/em = 690 nm) to find out the detection interval of its H₂O₂ concentration.

2.4 Detection of OPs by AZ NPs-ACh-sensor

ACh, a substrate for AChE, can be hydrolyzed by AChE to form choline, which can be further catalyzed by CHO to form H₂O₂. Therefore, the composition of the working fluid was determined based on experimental optimization. Firstly, AChE (0.8 U/mL) 10 μL and pesticide 5 μL were added to react for 10 min, after that, ACh (0.5 M) 50 μL was added, CHO (0.8 U/mL) 10 μL was reacted for 15 min, and then AZ NPs-ACh-sensor was added and incubated for 30 min. It was put into the enzyme marker (ex = 310 nm/em = 690 nm) for detection. The herbal samples were extracted by ultrasonic extraction for 3 min, then put into 4 °C for future use. 5 μL was added to react with AChE during the assay, after which the standardized operation was repeated.

3 Results and discussion

3.1 Synthesis of the AZ NPs-ACh-sensor

It was found that the ratio of HAuCl₄ and AgNO₃ had a significant effect on the synthesis of AuAg NCs under the same synthesis conditions. Therefore, the fluorescence intensity of the product was determined by adjusting different molar ratios of HAuCl₄ to AgNO₃, and ultimately a ratio of gold:silver of 4:1 was chosen (Zhou et al., 2016).

Using BSA to reduce Au and Ag precursors to metal monomers, AuAg NCs were synthesized with stable properties and high fluorescence intensities (Figure 1A). BSA was chosen as the reducing agent because the sulfur atoms within its proteins form coordination bonds with metal atoms to prevent metal aggregation. Meanwhile, it simultaneously participates in the reduction of metal ions, which effectively protects the stability and dispersion of nanoclusters and promotes the formation of nanoclusters. BSA also has abundant coordination sites that can coordinate with Zn²⁺ ions, and the histidine and cysteine residues within it can form stable Zn²⁺-N bonds, constituting a metal-protein complex for use as a fluorescent sensor (Lindskog, 1997; Tso et al., 2004; Majorek et al., 2012).

The morphology of this fluorescent sensor was characterized using High Angle Annular Dark Field Scanning Transmission Electron Microscopy (HAADF-STEM) (Figure 1B). Elemental facet distribution analysis by HAADF-STEM confirms that the elements Au and Ag (Figure 1C), N (Figure 1D), Zn (Figure 1E), are co-localized in the structure of the sensor, and thus the successful synthesis of the sensors can be proved the formation of fluorescent sensor. The particle size distribution was further validated by dynamic light scattering (DLS) analysis, revealing that AuAg NCs exhibited a size of approximately 5–10 nm with uniform morphology and dimensional stability (Figure 1F), while the average particle size of the sensor was about 400 nm (Figure 1G).

After fixing the amount of Zn(NO₃)₂ during the preparation of BSA-AuAg NCs into fluorescent sensors, it was observed that the fluorescence intensity of the nanocluster solution was enhanced with increasing volume in the range of 1–4 mL (Figure 1H). Therefore, 4 mL of BSA-AuAg NCs was chosen as the amount for subsequent sensor synthesis. The optimization of the reaction time (Figure 1I) and synthesis method (Figure 1J) using the control variable method was continued to determine the optimal synthesis scheme for the sensor.

3.2 Characterization of the AZ NPs-ACh-sensor

The UV-visible spectra of AuAg NCs and the AZ NPs-ACh-sensor were recorded from 240 to 700 nm using a UV spectrophotometer. As shown in (Figure 2A), their spectral profiles exhibited minimal change before and after sensor synthesis, confirming excellent optical stability. Full-wavelength scanning on a microplate reader revealed the best AZ NPs-ACh-sensor excitation and emission peaks at 310 nm and 690 nm, respectively (Figure 2B). Compared to the low Signal-to-Background Ratio (SBR) caused by short Stokes shifts, larger Stokes shifts (>380 nm) can avoid interference from the spontaneous luminescence of biological samples, making them suitable for the detection of complex samples and avoiding spectral overlap (Choi et al., 2006; Araneda et al., 2011; Guo et al., 2023).

Sensor stability is a critical prerequisite for reliable measurements. We systematically evaluated the AZ NPs-ACh-sensor performance under varying environmental conditions (temperature, pH, different polarity solution). Results indicate consistent optical responses within physiological pH (6.0–7.4) (Figure 2C) and temperature (30 °C–37 °C) range (Figure 2D), providing a basis for optimizing experimental conditions and practical applications. This stability originates from bovine serum BSA conformational integrity within the sensor structure. Under strong alkaline conditions, electrostatic repulsion between BSA molecules can irreversibly disrupt its tertiary structure, compromising the sensor’s physical integrity (Prasanthan and Kishore, 2019). Similarly, thermal denaturation at elevated temperatures may expose and aggregate nanoclusters originally stabilized by the protein, degrading optical performance (Timr et al., 2020). Therefore, the observed optimal stability window coincides with the physiological conditions under which the BSA protein maintains its functional conformation.

Polar stability was tested in methanol-water mixtures (Figure 2E). The sensor demonstrated high stability across varying solvent ratios, enabling applications in diverse detection systems regardless of solution polarity. Fluorescence stability assays over 15 days (Figure 2F) revealed significantly enhanced stability of the sensor compared to bare AuAg NCs. This improvement is attributed to stable Zn²⁺-N coordination bonds, which effectively suppress nanocluster aggregation and fluorescence quenching.

In the actual application environment often contains a variety of interfering substances, the sensor’s anti-interference ability is an important indicator to evaluate its performance. For this reason, the selectivity of the developed sensor was systematically examined in this study, and a variety of common metal ions and amino acids were selected as potential interferents for testing (Figure 2G). The experimental results show that the sensor exhibits a significant response signal only for H₂O₂, while there is no significant response for other test substances, indicating that it has good anti-interference ability and certain analytical specificity. This selective mechanism can be attributed to the ability of H₂O₂ to specifically disrupt the Zn²⁺-N coordination bond in the structure of the sensor, and the breakage of the bond leads to a significant reduction in the fluorescence emission of the sensor. As shown in (Figure 2H), at 310 nm excitation wavelength, the fluorescence emission intensity of the sensor at 690 nm showed an obvious decreasing trend after the addition of H₂O₂, which visually corroborated the above mechanism of action.

3.3 The AZ NPs-ACh sensor detects H₂O₂ concentration

The Zn²⁺-N bond in the sensor breaks in the presence of H₂O₂, leading to a fluorescence burst in the sensor (Hu et al., 2018; Jiang et al., 2021). In the low wavenumber region (500–400 cm^-1), a distinct absorption peak at approximately cm^-1 is attributed to the stretching vibration of the Zn²⁺-N bond. Following H₂O₂ treatment, the intensity of the Zn²⁺-N vibration significantly diminishes compared to that observed in untreated AZ NPs-ACh sensors (Supplementary Figure S1). This attenuation directly reflects H₂O₂’s potential to induce bond breakage or coordination environment reorganization within the AZ NPs framework. This structural alteration provides crucial molecular-level evidence for the interaction between H₂O₂ and the AZ NPs-ACh sensing element, underpinning the sensor’s response behavior to H₂O₂. XPS analysis revealed a distinct alteration in the chemical state of Zn following H₂O₂ treatment. As shown in (Supplementary Figure S2), the Zn 2p_3/2 peak was deconvoluted into two new components at approximately 1026 eV and 1048 eV. This change might be ascribed to the transformation of the Zn coordination environment from nitroge to oxygen ligands (Sandip et al., 2008; Cao et al., 2026). Notably, the sensor exhibits a wide range of excitation spectral response, a feature that offers the possibility of easy and intuitive H₂O₂ visualization. As shown in (Figure 3A), under the irradiation of 365 nm UV lamp, when the sensor reacts with a series of different concentrations of H₂O₂ solution, a continuous change of fluorescence intensity from strong to weak can be observed by the naked eye, showing a gradient from bright to dim. To further quantify this reaction, we performed the assay using an enzyme marker under optimized excitation 310 nm and emission 690 nm wavelength conditions. As shown in (Figure 3B), the experimental data clearly confirmed that there was a good correspondence between the change in fluorescence intensity and H₂O₂ concentration in the range of 0–10 mM.

In addition, the dynamic response characteristics of the sensor were also evaluated, as shown in (Figure 3C), where the sensor exhibited fast response kinetics upon reaction with H₂O₂, with a significant decrease in fluorescence intensity observed within 5 min. More importantly, the fluorescence intensity of the system was able to remain stable up to 60 min into the reaction, which provided for reliable kinetic monitoring. Further, we quantified the sensitivity of the sensor for detecting H₂O₂. As shown in (Figure 3D), by plotting the calibration curve between the fluorescence intensity change and H₂O₂ concentration, a good linearity (y = 0.92206-0.00214x) was confirmed in the concentration range of 5 μM–100 μM (Figure 3E), the LOD was determined to be 1.4 μM and LOQ was 4.7 μM. This demonstrates the ability of the sensor to accurately quantify low concentrations of H₂O₂.

3.4 Detection of AChE activity by the AZ NPs-ACh sensor

AChE is able to catalyze the hydrolysis of ACh highly specifically to produce choline (Ch) and AcOH. ChOx can further oxidize Ch to produce H₂O₂ as a downstream signaling molecule 15 (Cai et al., 2021; Wang et al., 2024). The final product of this cascade reaction, H₂O₂, is capable of interacting with the sensing interface and triggering a fluorescent signal burst. Based on this principle, we constructed a fluorescence sensing strategy for quantitative assessment of AChE activity, where the response signal is directly correlated with the amount of H₂O₂ production.

In order to systematically elucidate and validate the effectiveness of the above sensing mechanisms, we carried out a series of feasibility experiments. First, we prepared a mixed working solution containing AChE, ChOx, and the substrate acetylcholine chloride. As shown in (Figure 4A), the experimental results indicated that no significant fluorescence burst was observed for the sensor probe with only a single component in the working solution (i.e., AChE, ChOx or ACh). This observation strongly rules out the effect of non-specific interactions or background interference on the sensor signal readout. Furthermore (Figure 4B), when the working fluid contained only two components, no effective generation of H₂O₂ was detected, and thus no fluorescence quenching was induced. This result clearly confirms that the enzymatic reaction system does not possess the ability to produce the signaling molecule H₂O₂ in the absence of any enzyme or substrate, thus verifying that the substrate-induced cascade reaction in the proposed sensing mechanism is indispensable for the signal generation.

The follow-up experiments examined the effect of key component concentrations on sensor performance, as shown in (Supplementary Figures S3–S5). With other conditions fixed, the results showed that the fluorescence signal continued to decrease as the concentration of ChOx increased, and the fluorescence inhibition rate stabilized when the ChOx concentration reached 0.8 U/mL. Using the control variable method, it was further determined that the optimal AChE concentration was 0.8 U/mL and the optimal ACh concentration was 0.5 M, conditions under which the signal response was most favorable. Additionally, considering the property of H₂O₂ undergoing hydrolysis when exposed to high temperatures for extended periods, the experiment examined changes in H₂O₂ concentration over 40 min at different temperatures. The results indicate that under optimal enzyme conditions, the stability of H₂O₂ at various concentrations supports experimental procedures regardless of whether titanium sulfate detection or AZ NPs-ACh sensor detection was employed (Supplementary Figures S6 and S7).

The optimization of reaction time is equally critical. Experiments showed that the optimal incubation time for the sensor and the working solution was 30 min, at which time the enzymatic reaction was basically completed (Figure 4C). Meanwhile, the sensor’s fluorescence signal reached a steady state within 20 min after the end of incubation, which ensures the timeliness of the detection and the reliability of the results (Figure 4D). Through system optimization, the sensor exhibited good response characteristics and stability under specific conditions.

3.5 Detection of OPs by AZ NPs-ACh sensor

Triazophos, a class of broad-spectrum OPs, has been banned in numerous countries due to its inherent high toxicity and risk of persistent residues. In this study, we explored the quantitative detection of triazophos using a sensing system constructed on the principle of AChE inhibition (Jiang et al., 2009). The procedure was as follows: first, the triazophos standard was co-incubated with AChE for 15 min, followed by the addition of ACh and ChOx, with continued incubation for 30 min. Based on the specific inhibitory mechanism of OPs on AChE activity, the presence of triazophos would hinder the effective hydrolysis of ACh, which in turn would inhibit the downstream ChOx-catalyzed oxidation reaction, leading to insufficient generation of H₂O₂ in the system. Subsequently, a fluorescent sensor based on H₂O₂ response was introduced to indirectly reflect the concentration of triazophos by detecting the production of H₂O₂.

After incubation with the sensor, changes in fluorescence intensity were measured using a microplate reader at excitation and emission wavelengths of 310 nm and 690 nm, respectively. The experimental results demonstrated a good linear response between triazophos concentration (ranging from 2 ng/mL to 200 ng/mL, the LOD and LOQ were calculated as 0.69 ng/mL and 2.0 ng/mL) and relative fluorescence intensity (Figure 5A), confirming the feasibility of this sensing strategy for triazophos detection. The reliability of the method was verified through spiking and recovery tests using dried Chenpi samples (Figure 5B). Additionally, ten batches of Chenpi samples were tested to validate method applicability. As shown in Supplementary Table S1 and Supplementary Figure S8, rapid ice bath extraction during triazophos extraction minimizes interference from endogenous matrices. The results confirmed that this sensor is capable of detecting triazophos in complex matrices.

The sensor also exhibited multi-solvent tolerance, maintaining high fluorescence intensity in pure water, methanol, ethanol, ethyl acetate, and other solutions (Figure 5C). As a result, the sensor enables in-situ detection directly in extraction solutions of complex samples without the need for secondary transfer to specific solvents, significantly simplifying the pretreatment process. This feature allows even non-professionals to perform basic pretreatment procedures outside laboratory settings.

To further expand the application scenarios of sensing systems and enhance their practicality, we have achieved paper-based immobilization of sensing materials. This method enables more intuitive implementation of sensor applications (Wang et al., 2020; Pang et al., 2025).This paper-based material exhibits stability between 20 °C and 50 °C Celsius. When stored dry and protected from light within 3 days of preparation, its fluorescence intensity remains stable, demonstrating practical utility (Supplementary Figure S9). In the experiment, a solution containing the target analyte (represented by the signaling molecule H₂O₂) was dropped onto the paper-based substrate loaded with the sensing material (Figures 5D,E). Owing to the wide excitation spectrum of the sensing material, a significant fluorescence quenching phenomenon can be observed with the naked eye under simple illumination from a 365 nm ultraviolet lamp, decreasing as the H₂O₂ concentration increases (Supplementary Figure S10). This demonstrates the potential of the paper-based sensing device for preliminary screening of pesticide residues (exemplified by triazophos). The procedure can be simplified as follows: after co-incubating the sample with the enzyme reaction working solution, a suitable amount of the reaction solution is dropped onto the paper-based sensing zone, and fluorescence changes are observed under a UV lamp. This straightforward method provides a promising technical approach for on-site or rapid preliminary assessment of pesticide residue levels.

4 Conclusion

In this study, a novel organophosphorus pesticide (OPs) sensor, designated as the AZ NPs-ACh sensor, was successfully developed. The sensor utilizes AZ NPs coordinated with gold-silver nanoclusters (AuAg NCs) and Zn²⁺ ions to achieve detection functionality. By specifically capturing H₂O₂—a downstream signaling molecule of enzymatic reactions—and leveraging the inhibition mechanism of OPs on acetylcholinesterase (AChE), it enables highly efficient OPs analysis based on an enzyme-linked amplification strategy. Two major innovative features of the sensor: firstly, it demonstrates exceptional multi-solvent tolerance, maintaining over 95% fluorescence intensity stability in diverse environments including pure water, methanol, ethanol, and ethyl acetate, which is crucial for analyzing complex real-world samples; Secondly, the successful integration of sensing materials onto a paper-based platform enables direct observation of fluorescence quenching signals under a 365 nm portable UV lamp (such as a counterfeit detector pen), facilitating semi-quantitative detection of pesticide residues in farm fields and markets.Beyond its practical value, this study elucidates the synthetic mechanism of the sensor and establishes a new paradigm for convenient, user-friendly visual monitoring of pesticide residues. This is achieved by synergistically integrating solvent-tolerant sensing technology with a paper-based device design.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fsens.2026.1756022/full#supplementary-material

Supplementary material Supplementary material at the source

Footnotes

Edited by: Shi Gang Liu, Hunan Agricultural University, China

Reviewed by: Husam Abduldaem Mohammed, University of Baghdad, Iraq

Ashok Kumar, VIT University, India