A Rapid and Sensitive Gold Nanoparticle-Based Lateral Flow Immunoassay for Chlorantraniliprole in Agricultural and Environmental Samples.

Chlorantraniliprole (CAP) is a new type of diamide insecticide that is mainly used to control lepidopteran pests. However, it has been proven to be hazardous to nontarget organisms, and the effects of its residues need to be monitored. In this study, five hybridoma cell lines were developed that produced anti-CAP monoclonal antibodies (mAbs), of which the mAb originating from the cell line 5C5B9 showed the highest sensitivity and was used to develop a gold nanoparticle-based lateral flow immunoassay (AuNP-LFIA) for CAP. The visible limit of detection of the AuNP-LFIA was 1.25 ng/mL, and the detection results were obtained in less than 10 min. The AuNP-LFIA showed no cross-reactivity for CAP analogs, except for tetraniliprole (50%) and cyclaniliprole (5%). In the detection of spiked and blind samples, the accuracy and reliability of the AuNP-LFIA were confirmed by a comparison with spiked concentrations and verified by ultra-performance liquid chromatography-tandem mass spectrometry. Thus, this study provides the core reagents for establishing CAP immunoassays and a AuNP-LFIA for the detection of residual CAP.

Development of competitive and noncompetitive lateral flow immunoassays for pendimethalin using synthetic peptides.

Peptidomimetic and anti-immunocomplex peptides can be easily isolated from phage display libraries, and can be used as alternatives to chemical competing haptens to improve the sensitivity of small molecule immunoassay. In this work, 16 peptidomimetics and 7 anti-immunocomplex peptides of pendimethalin (PND) were obtained from cyclic 7-, 8-, 9-, and 10-residue peptide phage libraries. Peptidomimetic EJ-2 (CMFTGTDFPC) with the highest sensitivity in competitive phage enzyme-linked immunosorbent assay (ELISA) and immunocomplex peptide EF-30 (CNPGWPPIPC) with the highest sensitivity in noncompetitive phage ELISA were selected to prepare phage-free peptides with GGGSSK-biotin at the C-terminus. Competitive and noncompetitive lateral flow immunoassays (CLFIA and NLFIA) were developed by using the phage-free peptides. After optimization, the CLFIA and NLFIA showed visual limit of detections (vLODs) of 5 ng/mL and 2.5 ng/mL, respectively, which were improved two- and fourfold compared with a LFIA based on chemical hapten. The NLFIA showed better sensitivity than CLFIA in the detection of spiked samples, and can meet the detection requirements for agro-products regulated by EU and China. The detection results of CLFIA and NLFIA for blind samples were consistent with that of ultra performance liquid chromatography/tandem mass spectrometry.

Monoclonal Antibody-Based Colorimetric Lateral Flow Immunoassay for the Detection of Pyridaben in the Environment.

Pyridaben, a broad-spectrum pyridazinone acaricide that is widely used in agricultural production, can induce neurotoxicity and reproductive abnormalities, and is highly toxic to aquatic organisms. In this study, a pyridaben hapten was synthesized and used to prepare monoclonal antibodies (mAbs), among which 6E3G8D7 showed the highest sensitivity in indirect competitive enzyme-linked immunosorbent assay, with a 50% inhibitory concentration (IC50) of 3.49 ng mL-1. The mAb, 6E3G8D7, was further applied to a gold nanoparticle-based colorimetric lateral flow immunoassay (CLFIA) for pyridaben detection, according to the signal intensity ratio of the test line to the control line, which showed a visual limit of detection of 5 ng mL-1. The CLFIA also showed high specificity and achieved excellent accuracy in different matrices. In addition, the amounts of pyridaben in blind samples detected by the CLFIA, were consistent with high-performance liquid chromatography. Therefore, the developed CLFIA is considered a promising, reliable, and portable method for pyridaben on-site detection in agro-products and environmental samples.

A Monoclonal Antibody-Based Immunochromatographic Test Strip and Its Application in the Rapid Detection of Cucumber Green Mottle Mosaic Virus.

Two specific monoclonal antibodies (mAbs) were screened, and an immunochromatographic strip (ICS) test for rapid and specific detection of cucumber green mottle mosaic virus (CGMMV) was developed. The coat protein of CGMMV was heterologously expressed as an immunogen, and specific capture mAb 2C9 and the detection mAb 4D4 were screened by an uncompetitive immunoassay. The test and control lines on the nitrocellulose membrane were coated with the purified 2C9 and a goat anti-mouse IgG, respectively, and a nanogold probe combined with 4D4 was applied to the conjugate pad. Using these mAbs, a rapid and sensitive ICS was developed. Within the sandwich mode of 2C9-CGMMV-4D4, the test line showed a corresponding positive relationship with CGMMV in infected samples. The ICS test had a detection limit of 1:5000 (w/v) for CGMMV in samples and was specific for CGMMV, with no observed cross-reaction with TMV or CMV.

Rapid and sensitive detection of quizalofop-p-ethyl by gold nanoparticle-based lateral flow immunoassay in agriproducts and environmental samples.

Quizalofop-p-ethyl is a widely used herbicide that poses a threat to human health and environmental safety. In this study, anti-quizalofop-p-ethyl monoclonal antibodies (mAbs) were prepared and used to develop a gold nanoparticle-based lateral flow immunoassay (AuNP-LFIA) for the detection of quizalofop-p-ethyl in agriproducts and environmental samples. Four hybridoma cell lines were obtained, among which 5B6D10E11 secreted mAb with the highest sensitivity, with a 50 % inhibition concentration of 4.57 ng/mL in the indirect competitive enzyme-linked immunosorbent assay. After optimization, the AuNP-LFIA strip based on the mAb (5B6D10E11) showed a visual detection limit of 10 ng/mL, and the results could be directly determined by the naked eye within 8 min. The cross-reactivity of the AuNP-LFIA for analogs of quizalofop-p-ethyl was negligible except for quizalofop-p-acid. The established AuNP-LFIA was proven to be accurate and precise based on the recovery test. Furthermore, the detection results of AuNP-LFIA were consistent with those of ultra-high-performance liquid chromatography tandem mass spectrometry.

Development of Ic-ELISA and Colloidal Gold Lateral Flow Immunoassay for the Determination of Cypermethrin in Agricultural Samples.

Cypermethrin (CYP) is an insecticide in the pyrethroid family and is used widely in agriculture and for public health purposes. However, CYP has been shown to have negative impacts on reproduction, immunity and nerves in mammals. In this study, a monoclonal antibody (mAb) against CYP was prepared and used to establish an indirect competitive immunosorbent assay (ic-ELISA) and colloidal gold lateral flow immunoassay (LFIA) for the quantitative and qualitative determination of CYP residues in agricultural products. The half inhibition concentration of the ic-ELISA was 2.49 ng/mL, and the cut-off value and visual limit of detection of the LFIA were 0.6 and 0.3 µg/mL, respectively. The recovery rates of the ic-ELISA ranged from 78.8% to 87.6% in tomato, cabbage and romaine lettuce. The qualitative results of LFIA and quantitative results of ic-ELISA and HPLC were in good agreement in blind samples. Overall, the established ic-ELISA and LFIA proved to be accurate and rapid methods for the determination of CYP in agricultural products.

Biotin-labelled peptidomimetic for competitive time-resolved fluoroimmunoassay of benzothiostrobin.

In recent years, more and more functional peptide ligands have been identified from phage display libraries and served the immunoassay of small molecules. After the identification, the phage particle instead limits further application of peptide ligands, so it is of great significance to explore the peptide ligand as an independent detection reagent. In this work, the identified peptidomimetic of benzothiostrobin was synthesized and labelled with biotin, which was combined with Eu3+-labelled streptavidin to develop the peptide-based time-resolved fluoroimmunoassay (P-TRFIA). Under the optimal conditions, the half-maximum inhibitory concentration (IC50) of proposed P-TRFIA is 3.63 ng mL-1, which is similar to the TRFIA using phage-borne peptidomimetic and Eu3+-labelled anti-phage antibody (IC50: 4.55 ng mL-1), also more sensitive than previously reported immunoassays for benzothiostrobin. In addition, the proposed P-TRFIA shows excellent specificity and accuracy for analysis of spiked samples, and its detection results shows good consistency with high-performance liquid chromatography for the detection of environment and agro-products samples with unknown benzothiostrobin concentrations.

New Approach to Generate Ratiometric Signals on Immunochromatographic Strips for Small Molecules.

The self-calibration capability of ratiometric signals has been widely considered to enhance the accuracy, sensitivity, and anti-interference ability of immunoassays. Exploring a new approach to generate ratiometric signals can provide more options for various requirements. Herein, we integrated the negative-readout competitive and positive-readout noncompetitive immunoassays into a single assay by employing different color tracers, labeled peptidomimetic and anti-immunocomplex peptides, to create a new unconstrained ratiometric signal approach. Using an immunochromatographic strip (ICS) and a fungicide benzothiostrobin as the analytical platform and analyte, respectively, we showed that this approach can be extensively applied to fluorescence and colorimetry readouts, which have also been proven for strong anti-interference ability to an external light environment. Moreover, the enormous intuitional color changes of ratiometric fluorescent and colorimetric ICSs (RFICS and RCICS) enabled the formation of the color reference cards (like the pH paper) for visual judgment. After adaptation with a portable smartphone, the quantitative detection limits for RFICS and RCICS were 0.17 and 0.44 ng mL-1, respectively. In addition, the ICSs showed good accuracy for the detection of benzothiostrobin in spiked samples.

Development of immunoassay based on rational hapten design for sensitive detection of pendimethalin in environment.

Pendimethalin (PND) is one of the most widely used selective herbicides, but it is considered a potential human carcinogen and persistent bioaccumulative toxic chemical. Herein, five haptens with carboxylic groups were synthesized based on rational design and used to immunize mice, respectively. Then the antibodies obtained were evaluated systematically, and an indirect competitive ELISA (ic-ELISA) was developed based on an anti-PND monoclonal antibody. The 50% inhibition concentration and limit of detection of ic-ELISA were 0.53 ng/mL and 0.07 ng/mL, respectively. The cross-reactivities of ic-ELISA for the analogs of PND were ≤ 1.1%. The average recoveries of PND ranged from 79.5% to 107.4% in spiked samples. A good correlation was achieved between the ic-ELISA results and UPLC-MS/MS results in the analysis of blind samples. Thus, this assay provides a rapid and accurate tool for the determination of PND in the agro-products and agricultural producing environment.

"Ready-to-use" immunosensor for the detection of small molecules with fast readout.

Immunoassays are commonly used methods for detection of small molecules that typically require numerous steps of the labeling between immune-recognition reagents and tracers, immobilization and recurrent washing, making them time consuming and difficult to adapt into point of care formats. Here we describe a "ready-to-use" homogeneous competitive immunosensor with an assay time of 10 min that is based exclusively on recombinant reagents. The signal is produced when the split fragments of the nano luciferase (Nluc) are brought together by the interaction of a heavy chain only variable domain (VHH) with a peptidomimetic of the target small molecule. A VHH to 2,4-dichlorophenoxyacetic acid (2,4-D) was used to isolated the peptidomimetic (NGFFEPWQVVYV) from phage display libraries using six panning conditions. Then the peptidomimetic and VHH were fused with the larger (LgN) and smaller piece (SmN) of split fragments of Nluc, respectively. In order to optimize the signal and sensitivity of the immunosensor, we explored the effects of the spacer between the peptidomimetic and LgN, the copy number of peptidomimetics, and the spacer between SmN and VHH, generating 24 combinations that allowed to conclude on their respective roles. Eventually, the developed "ready-to-use" immunosensor performed excellent signal-to-noise ratio and sensitivity, and could be applied to the detection of 2,4-D in real samples. Meanwhile, the immunosensor totally realizes labeling-free, immobilization-free and washing-free, also can be produced in a highly cost effective way.

Identification and Application of Two Promising Peptide Ligands for the Immunodetection of Imidacloprid Residue.

As the most widely used neonicotinoid insecticide, it is of great significance to explore the immunoreagents and immunoassays for imidacloprid (IMI) residue. In immunoassays, specific peptide ligands, such as peptidomimetic and anti-immunocomplex peptides, are regarded as promising substitutes for chemical haptens. In the present work, we identified thirty sequences of peptidomimetics and two sequences of anti-immunocomplex peptides for IMI from three phage pVIII display cyclic peptide libraries, in which the anti-immunocomplex peptides are the first reported noncompetitive reagents for IMI. The peptidomimetic 1-9-H and anti-immunocomplex peptide 2-1-H that showed the best sensitivity were utilized to develop competitive and noncompetitive phage enzyme-linked immunosorbent assays (P-ELISAs), with a half inhibition concentration of 0.55 ng/mL for competitive P-ELISA and a half-saturation concentration of 0.35 ng/mL for noncompetitive P-ELISA. The anti-immunocomplex peptide was demonstrated to greatly improve the specificity compared with competitive P-ELISA. In addition, the accuracy of proposed P-ELISAs was confirmed by recovery analysis and HPLC verification in agricultural and environmental samples. These results show that the peptide ligands identified from phage display library can replace chemical haptens in the immunoassays of IMI with the satisfactory performance.

Electrofusion preparation of anti-triazophos monoclonal antibodies for development of an indirect competitive enzyme-linked immunosorbent assay.

Immunoassays have been widely used to detect small molecular contaminants due to the advantages of simplicity, high throughout and low-cost. Antibodies are essential reagents of immunoassays, their quality directly determines the characteristics of immunoassays. In this study, the monoclonal antibodies (mAbs) of triazophos were prepared by electrofusion, and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Under the optimal electrofusion conditions (cells treatment with pronase, the alternating electric field strength of 45 V cm-1, the direct current voltage of 3 kV), the fusion efficiency was 1.104 ± 0.063‱, which was improved more than 4-fold compared with the chemical fusion method (0.255 ± 0.089‱). Three hybrid cell lines that can stably secrete the anti-triazophos mAbs were obtained. The cell line 4G6F10 showed the highest sensitivity, which was used to generate mAb and develop an ic-ELISA. After optimization, the 50% inhibition concentration (IC50), limit of detection (LOD) and linear range (IC10-IC90) of the ic-ELISA were 0.32 ng mL-1, 0.08 ng mL-1 and 0.08-2.17 ng mL-1, respectively. There was no significant cross-reactivity with the analogues of triazophos. The average recoveries of triazophos in spiked samples were 77.5%-89.3% with the relative standard deviations of 0.1%-9.2%. In addition, the ic-ELISA showed good repeatability, reproducibility and accuracy for the analysis of apple samples spiked with triazophos.

Development of competitive and noncompetitive immunoassays for clothianidin with high sensitivity and specificity using phage-displayed peptides.

Clothianidin is a widely used pesticide that has been banned from outdoor use by the European Union due to its toxicity. To improve the sensitivity and specificity of existing clothianidin immunoassays, we developed competitive and noncompetitive immunoassays for clothianidin based on phage-displayed peptides. Cyclic 8-, 9-, and 10-residue peptide libraries were constructed using an optimized phagemid pComb-pVIII to prevent the loss of theoretical library diversity. Twenty-eight peptidomimetics and two anti-immunocomplex peptides were isolated through a blended panning process and used to develop competitive and noncompetitive phage enzyme-linked immunosorbent assays (P-ELISAs), respectively. After optimization, the half inhibition concentration (IC50) and half saturation concentration (SC50) of competitive and noncompetitive P-ELISAs were 3.83 ± 0.23 and 0.45 ± 0.02 ng/mL, respectively. Competitive P-ELISA showed 2.6-18.2% cross-reactivity with imidaclothiz, nitenpyram and imidacloprid. Importantly, noncompetitive P-ELISA, which has the best specificity and great sensitivity for clothianidin, showed no cross-reactivity with the analogs. The average recoveries of competitive and noncompetitive P-ELISAs were 73.8-104.1% and 76.6-102.2%, respectively, while the relative standard deviations were ≤ 11.0%. In addition, the results of P-ELISAs in the analysis of blind samples were consistent with those of high-performance liquid chromatography.

Intrinsic Clearance and Metabolism Pathway of Fosthiazate in Rat and Cock Liver Microsomes: From Chiral Assessment View.

Chiral fosthiazate enters the organisms via environmental exposure and food web enrichment. Liver subcellular fractions of rats (RLM) and cocks (CLM) were prepared to explore the stereoselective metabolism of fosthiazate in vitro. The results indicated that fosthiazate exhibited different stereoselective metabolism behaviors in RLM and CLM. The clearance rate order of RLM to four fosthiazate stereoisomers was (1R,3R)-fosthiazate > (1S,3R)-fosthiazate > (1R,3S)-fosthiazate > (1S,3S)-fosthiazate. However, CLM showed a faster clearance rate to (1S,3S)-fosthiazate and (1S,3R)-fosthiazate than the other two stereoisomers. The molecular docking results revealed that the stereoselectivity was partially due to the stereospecific binding between fosthiazate stereoisomers and cytochrome P450 proteins. The main metabolism pathways of fosthiazate in RLM and CLM were oxidation and hydrolysis with five common metabolites including M299, M243, M227, M103, and M197 being identified by LC-TOF-MS/MS. The present study provides the accurate data on risk assessment of chiral fosthiazate.

Competitive immune-nanoplatforms with positive readout for the rapid detection of imidacloprid using gold nanoparticles.

Two high-sensitivity competitive immune-nanoplatforms based on the inner filter effect (IFE-IN) and magnetic separation (MS-IN) with a positive readout were developed to rapidly detect imidacloprid (IMI) using gold nanoparticles (AuNPs). For IFE-IN, IMI competes with AuNPs-labeled IMI antigens (IMI-BSA-AuNPs) to bind with anti-IMI monoclonal antibody (mAb)-conjugated NaYF4:Yb,Er upconversion nanoparticles, which changes the fluorescence signal at excitation/emission wavelength of 980/544 nm. For MS-IN, the immunocomplex of IMI-BSA-AuNPs and magnetic-nanoparticles-labeled mAb (mAb-MNPs) dissociates in the presence of IMI, and the optical density of IMI-BSA-AuNPs at 525 nm increases with the IMI concentration after magnetic separation. Under the optimal conditions, the IMI concentration producing a 50% saturation of the signal (SC50) and linear range (SC10- SC90) were found to be 4.30 ng mL-1 and 0.47 - 21.37 ng mL-1 for IFE-IN, while 1.21 ng mL-1 and 0.07 - 10.21 ng mL-1 for MS-IN, respectively. Both IFE-IN and MS-IN achieved excellent accuracy for the detection of IMI in different matrices. The quantities of IMI in apple samples detected by IFE-IN and MS-IN were consistent with the high-performance liquid chromatography results. For IFE-IN, analyte competes with AuNPs-labeled-antigen to bind with the mAb-conjugated-UCNPs, which changes the fluorescence signal at 544 nm. For MS-IN, the immunocomplex of AuNPs-labeled-antigen and mAb-conjugated-MNPs dissociates in the presence of analyte, and the optical density of AuNPs-labeled-antigen at 525 nm increases with increasing analyte concentration after separation.

Sortase-Mediated Phage Decoration for Analytical Applications.

Phage-borne peptides and antibody fragments isolated from phage display libraries have proven to be versatile and valuable reagents for immunoassay development. Due to the lack of convenient and mild-condition methods for the labeling of the phage particles, isolated peptide/protein affinity ligands are commonly removed from the viral particles and conjugated to protein tracers or nanoparticles for analytical use. This abolishes the advantage of isolating ready-to-use affinity binders and creates the risk of affecting the polypeptide activity. To circumvent this problem, we optimized the phage display system to produce phage particles that express the affinity binder on pIII and a polyglycine short peptide fused to pVIII that allows the covalent attachment of tracer molecules employing sortase A. Using a llama heavy chain only variable domain (VHH) against the herbicide 2,4-D on pIII as the model, we showed that the phage can be extensively decorated with a rhodamine-LPETGG peptide conjugate or the protein nanoluciferase (Nluc) equipped with a C-terminal LPETGG peptide. The maximum labeling amounts of rhodamine-LPETGG and Nluc-LPETGG were 1238 ± 63 and 102 ± 16 per phage, respectively. The Nluc-labeled dual display phage was employed to develop a phage bioluminescent immunoassay (P-BLEIA) for the detection of 2,4-D. The limit of detection and 50% inhibition concentration of P-BLEIA were 0.491 and 2.15 ng mL-1, respectively, which represent 16-fold and 8-fold improvement compared to the phage enzyme-linked immunosorbent assay. In addition, the P-BLEIA showed good accuracy for the detection of 2,4-D in spiked samples.

Competitive and noncompetitive fluorescence polarization immunoassays for the detection of benzothiostrobin using FITC-labeled dendrimer-like peptides.

Peptides obtained from phage display libraries are valuable reagents for small-molecule immunoassays. However, their application in fluorescence polarization immunoassays (FPIAs) is limited by phage particles. Here, monomer, dendrimer-like dimer, tetramer peptidomimetic and anti-immunocomplex tracers were designed and synthesized using lysine as special scaffolds and spacers to develop competitive and noncompetitive FPIAs for benzothiostrobin. The affinity between tracers and monoclonal antibodies or immunocomplexes increased with the tracer valence. A higher signal-to-noise ratio and sensitivity could be generated in the FPIAs based on tetramer tracers. The sensitivities of competitive (50% inhibitory concentration) and noncompetitive (50% saturation concentration) FPIAs were 19.71 ± 4.65 and 40.43 ± 2.73 ng mL-1, respectively. The spiked recoveries were 78.3%-105.2% with relative standard deviations (RSDs) of 0.7%-15.4% for the competitive FPIA, while 78.7%-115.3% with RSDs of 0.7%-12.5% for the noncompetitive FPIA. The amounts of benzothiostrobin in rice detected by the FPIAs were consistent with those detected by high performance liquid chromatography.

Highly specific nanobody against herbicide 2,4-dichlorophenoxyacetic acid for monitoring of its contamination in environmental water.

2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, is a small organic chemical pollutant in the environment. To develop a nanobody-based immunoassay for monitoring trace levels of 2,4-D, a step-wise strategy for the generation of nanobodies highly specific against this small chemical was employed. Firstly, we synthesized three novel haptens mimicking 2,4-D and assessed their influence on the sensitivity and specificity of the existing antibody-based assay. Polyclonal antibodies (pAb) from rabbits showed good sensitivity and moderate specificity for 2,4-D, pAb from llama based on selected haptens showed similar performance when compared to those from rabbits. Secondly, nanobodies derived from llama were generated for 2,4-D by an effective procedure, including serum monitoring and one-step library construction. One nanobody, NB3-9, exhibited good sensitivity against 2,4-D (IC50 = 29.2 ng/mL) had better specificity than the rabbit pAb#1518, with no cross-reactivities against the 2,4-D analogs tested. Thirdly, one-step fluorescent enzyme immunoassay (FLEIA) for 2,4-D based on a nanobody-alkaline phosphatase (AP) fusion was developed with IC50 of 1.9 ng/mL and a linear range of 0.4-8.6 ng/mL. Environmental water samples were analyzed by FLEIA and LC-MS/MS for comparison, and the results were consistent between both methods. Therefore, the proposed step-wise strategy from hapten design to nanobody-AP fusion production was successfully conducted, and the resulting nanobody based FLEIA was demonstrated as a convenient tool to monitor 2,4-D residuals in the environment.

Development of Fluorescence Polarization Immunoassay for Imidacloprid in Environmental and Agricultural Samples.

A fluorescence polarization immunoassay (FPIA) for the determination of imidacloprid (IMI) was developed with advantages of simple operation and short assay time. The haptens of IMI, acetamiprid (ACE), and thiamethoxam (THI) were conjugated with fluorescein isothiocyanate ethylenediamine (EDF) and 4'-Aminomethyl fluorescein (AMF), respectively, to prepare six fluorescence tracers. The conjugation of IMI hapten and EDF (IMI-EDF) was selected to develop the FPIA due to the largest fluorescent polarization value increase in the presence of anti-IMI monoclonal antibody. Under the optimum condition, the limit of detection, 50% inhibition concentration and detection range of the FPIA were 1.7, 4.8, and 1.7-16.3 µg/L, respectively. The cross-reactivities (CRs) with the analogs of IMI were negligible except for imidaclothiz with CR of 79.13%. The average recovery of spiked paddy water, corn and cucumber samples were 82.4-118.5% with the RSDs of 7.0-15.9%, which indicated the FPIA had good accuracy. Thus, the developed FPIA was a potential tool for the rapid and accurate determination of IMI in agricultural and environmental samples.

Development of potent inhibitors of the human microsomal epoxide hydrolase.

Microsomal epoxide hydrolase (mEH) hydrolyzes a wide range of epoxide containing molecules. Although involved in the metabolism of xenobiotics, recent studies associate mEH with the onset and development of certain disease conditions. This phenomenon is partially attributed to the significant role mEH plays in hydrolyzing endogenous lipid mediators, suggesting more complex and extensive physiological functions. In order to obtain pharmacological tools to further study the biology and therapeutic potential of this enzyme target, we describe the development of highly potent 2-alkylthio acetamide inhibitors of the human mEH with IC50 values in the low nanomolar range. These are around 2 orders of magnitude more potent than previously obtained primary amine, amide and urea-based mEH inhibitors. Experimental assay results and rationalization of binding through docking calculations of inhibitors to a mEH homology model indicate that an amide connected to an alkyl side chain and a benzyl-thio function as key pharmacophore units.