Recognition mechanism and sequence optimization of organophosphorus pesticides aptamers for better monitoring contaminations in food

Aptamers as a kind of biological recognition element have shown great potential in monitoring and the rapid quantification of organophosphorus pesticides(OPPs). However, molecules of OPPs are structurally similar and original aptamers selected by systematic evolution of ligands by exponential enrichment are usually long-chain bases, which hamper the further application under OPPs-aptamer recognition. The aim of the research was to develop a new strategy to design oligonucleotide sequences for binding OPPs by combination of experimental and molecular modeling methods. 3D models of aptamers binding OPPs were constructed, and binding energy and the most probable binding site for the OPPs were then determined by molecular docking, and the binding sites were further confirmed by the results of 2-AP replaced experiments. Based on the docking results, a new aptamer for detection 4 representative OPPs with only 29 bases was designed by reasonable truncation and mutation of the reported aptamer(named S4-29). The interaction between this new aptamer and OPPs were analyzed by molecular docking, microscale thermophoresis, circular dichroism and fluorometric analysis. The results revealed that the new aptamer exhibit more superior recognition performance to OPPs, which can be promote the monitoring ability of OPPs contaminations in food.

Computational interaction analysis of organophosphorus pesticides with different metabolic proteins in humans

Pesticides have the potential to leave harmful effects on humans, animals, other living organisms, and the environment. Several human metabolic proteins inhibited after exposure to organophosphorus pesticides absorbed through the skin, inhalation, eyes and oral mucosa, are most important targets for this interaction study. The crystal structure of five different proteins, PDBIDs： 3LII, 3NXU, 4GTU, 2XJ1 and 1YXA in Homo sapiens （H. sapiens）, interact with organophosphorus pesticides at the molecular level. The 3-D structures were found to be of good quality and validated through PROCHECK, ERRAT and ProSA servers. The results show that the binding energy is maximum -45.21 relative units of cytochrome P450 protein with phosmet pesticide. In terms of H-bonding, methyl parathion and parathion with acetylcholinesterase protein, parathion, methylparathion and phosmet with protein kinase C show the highest interaction. We conclude that these organophosphorus pesticides are more toxic and inhibit enzymatic activity by interrupting the metabolic pathways in H. sapiens.

Carboxylic Esterase and Its Associations With Long-term Effects of Organophosphorus Pesticides

To examine a） the effect of organophosphorus pesticide exposure on activity of carboxylic esterases, namely butyrylcholinesterase （BChE）, carboxylesterase （CarbE） and paraoxonase （PonE）; and b） the association of polymorphisms of BChE and PonE with individual genetic susceptibility to organophosphorus pesticide exposure. Methods A cross-sectional study was conducted in 75 workers exposed to organophosphorus pesticides and 100 non-exposed controls. The serum activity of these enzymes was measured. Variant forms of BCHE-K, PON-192, and PON-55 were detected. A symptom score was developed as a proxy measure of clinical outcomes. Results Activities of both BChE and CarbE were lower in exposed workers （27.3±21.65 runol.hl.mL^-l and 235.6±104.03 nmol-min^-l.mL^-l） than in non-exposed workers （78.313±30.354 nmol.h^-l.mL^-1 and 362.681_＋194.997 nmol.min^-1.mL^-1）. The activity of PonE was not associated with exposure status. The AChE activity in the exposed workers with BCHE-K genotype UU （61 cases）, genotype UK （12 cases） and genotype KK （2 cases） was 105.05, 84.42 and 79.00 mmol-h^-1.mL^-1, respectively and the accumulative symptom scores were 3.74, 9.17, and 12.50 accordingly. The AChE activity in the exposed workers with PON-192 genotype BB （37）, genotype AB （27） and genotype AA （11） was 116.8, 91.2, and 72,3 mmol-h^-1.mL^-1, respectively and the symptom scores were 2.00, 6.74, and 9.73 accordingly. The AChE activity in those with PON-55 genotype LL （70） and genotype LM （5） was 102.4 and 82.8 mmol-h^-1.mL^-1 and the symptom scores were 4.53 and 9.20. The symptom score was the highest in individuals with abnormal homozygote for each of the three gene loci. Condusions Long-term exposure to organophosphorus pesticides can inhibit BChE and CarbE activity, but exerts no inhibitory effect on PonE activity. Different genotypes of BCHE-K, PON-192, and PON-55 may be related to the severity of adverse health effects of organophosphorus pesticide exposure. Implications of potentially higher susceptibility of workers with mutant homozygotes should be evaluated to reduce health risks.

Application of Current Hapten in the Production of Broad Specificity Antibodies Against Organophosphorus Pesticides

Diethylphosphono acetic acid （DPA） was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method （AEM） or 1-ethyl-3-（3-dimethylaminopropyl）-carbodimide method （EDC）. The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay （CI-ELISA）. A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL^-1and an I50 value of 0.182 μg mL^-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 μg mL^-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semiquantitation determination of the above former five organophosphorus pesticides.

Study of multiresidue analytical method for organonitrogen and organophosphorus pesticides in soil and water

A gas chromatographic method without derivatization was developed for the residue analysis of 10 organonitrogen and 9 organophosphorus pesticides in soil and water. The samples were blended or shaken with acetone for extraction. The extracts were cleaned up by coagulation, then, re-extracted with three 50 ml portions of dichloromethane. The final residue was detected by gas chromatography equipped with NPD. All of the 19 pesticides were completely separated at a constant temperature. The method described above was applicable to the simultaneous determination of 10 organonitrogen and 9 organophosphorus pesticides .in soil and water with the satisfactory recovery (from 82.42% to 103.57%), coefficient of variance (from 0.17% to 12.57%) and limit of detection (from 0.0006 ppm to 0.058 ppm).

Disposable Amperometric Acetylcholinesterase Biosensor for the Detection of Organophosphorus Pesticides

A rapid,simple,disposable and inexpensive acetylcholinesterase (ACHE) amperometric biosensor for the detection of organophosphorus pesticides was developed by simple adsorption of the enzyme on screen-printed electrodes.The biosensor consisted of an Ag/AgCl reference electrode and a graphite working electrode.The mixture of graphite and the 7,7,8,8-tetracyanoquinodimethane (TCNQ) was printed on electrodes.The detection of organophosphorus pesticides was done with acetylthiocholine chloride (ATCh) as substrate.The biosensor was used to detect the inhibitory effect of organophosphorus pesticides on AChE activity.The 1μl of enzyme solution containing 0.1 U AChE and 1% bovine serum albumin (BSA) were simply dropped on the working electrode surface.The biosensor operated at a potential of 300 mV vs. Ag/AgCl in a pH 7.2 0.1 mol/L phosphate buffer and 0.1 mol/L KCl.We obtained a calibration plot of the percentage inhibition versus the logarithm of parathion methyl concentration following an incubation time of 10 mix in parathion methyl solution. The lowest detectable amount of parathion methyl was 0.026 ppm.The amperometric biosensor based on acetylcholinesterase was disposable and low cost (about 1 yuan RMB).

Recent Advances in Degradation of Organophosphorus Pesticides

With the growing demand for environmental protection and physical health,food safety is now receiving more and more attention all over the world.However,pesticides are indispensable in agricultural production.Therefore,how to efficiently degrade pesticides and remove their residues in foods has always been a hot research topic in recent decades.This paper not only summarizes the types,degradation mechanism and artificial degradation of organophosphorus pesticides,but also highlights the latest advances in chemical degradation,photocatalytic degradation and biodegradation.

Visible light assisted enzyme-photocatalytic cascade degradation of organophosphorus pesticides

The worldwide application of organophosphorus pesticides(OPs)has promoted agricultural development,but their gradual accumulation in soil and water can seriously affect the central nervous system of humans and other mammals.Organophosphorus hydrolase(OPH)is an effective enzyme that can catalyze the degradation of the residual OPs.However,the degradation products such as p-nitrophenol(p-NP)is still toxic.Thus,it is of great significance to develop a multi-functional support that can be simultaneously used for the immobilization of OPH and the further degradation of p-NP.Herein,a visible light assisted enzyme-photocatalytic integrated catalyst was constructed by immobilizing OPH on hollow structured Au-TiO_(2)(named OPH@H-Au-TiO_(2))for the degradation of OPs.The obtained OPH@H-Au-TiO_(2)can degrade methyl parathion to p-NP by OPH and then degrade p-NP to hydroquinone with low toxicity by using H-Au-TiO_(2)under visible light.OPH molecules were immobilized on HAu-TiO_(2)through adsorption method to prepare OPH@H-Au-TiO_(2).After 2.5 h of reaction,methyl parathion is completely degraded,and about 82.64%of the generated p-NP is further degraded into hydroquinone.After reused for 4 times,the OPH@H-Au-TiO_(2)retains more than 80%of the initial degradation activity.This research presents a new insight in designing and constructing multi-functional biocatalyst,which greatly expands the application scenarios and industrial value of enzyme catalysis.

A sensitive and rapid UV-vis spectrophotometry for organophosphorus pesticides detection based on Ytterbium(Yb3+) functionalized gold nanoparticle

A kind of sensitive, rapid, and simple spectrophotometry based on a Ytterbium(Yb3+) functionalized gold nanoparticle(Au NPs-Yb) was developed for detection of organophosphorus pesticides(OPs). Prepared AuNPs possess oxygen-containing functional groups and strong complexation reaction with Yb3+. While oxygen-containing thiophosphate in the OPs molecule can combine with Yb3+as a cross-linking molecule to produce insoluble yetterbium phosphate, resulting in the aggregation of AuNPs and great decrease in ultraviolet absorbance strength at 520 nm by ultraviolet visible(UV-vis) spectrophotometer. Under the optimized conditions, a linear relationship between the absorbance of AuNPs and OPs concentration ranged from 0.05 μg/L to 6.0 μg/L with limit of detection for 0.03 μg/L(S/N = 3), which is far lower than the maximum residue limit(0.01 ppm) in the European Union pesticides database. Therefore, this assay has potential application in the determination of OPs in the field of environmental and food monitoring.

3DRGO-NiFe2O4/NiO nanoparticles for fast and simple detection of organophosphorus pesticides

The residues of organophosphorus pesticide(OPs)on fruits and vegetables pose a threat to human health,so it is very meaningful to explore simple and fast detect methods for OPs residual.In this work,nickel ferrite/nickel oxide nanoparticles co-loaded three-dimensional reduced graphene oxide(3DRGONiFe2O4/NiO NPs),as a new low cost nanocomposite,was prepared.Based on its high performance mimetic peroxidase activity,a colorimetric method for the detection of OPs has been developed.Dichlorvos was chosen as model compounds to evaluate the detection performance.The detection linear range for dichlorvos is from 50μg/mL to 2.5×10^4μg/mL with a detection limit of 10μg/mL.Furthermore,a test paper can be developed based on the 3 DRGO-NiFe2O4/NiO NPs for visual detection of dichlorvos,and the image information of the paper sensor can be converted into digital signal and quantitative detection by a smartphone.Notably,this method can also be used to detect dichlorvos in real samples,including vegetables and fruits.Thus,the developed naked assay holds great potential in simple,inexpensive and rapid detection of OPs in fruit and vegetable samples.

Separation of chlorinated hydrocarbons and organophosphorus, pyrethroid pesticides by silicagel fractionation chromatography and their simultaneous determination by GC-MS

A silicagel fractionation procedure for environmental sample extracts, which separates chlorinated hydrocarbons(CHCs) and organophosphorus, pyrethroid pesticides into two groups for subsequent instrumental analysis, was developed in this study. This method was achieved by optimizing the fraction cut-off volume of elution and different solvents. Using fully activated silica gel and cut-off CHCs collection after 10 ml 10% dichloromethane (DCM) in n-hexane passing through the column resulted in satisfactory separation of CHCs and organophosphorus, pyrethroid pesticides. This procedure had a higher reliability for CHCs than for organophosphorus, pyrethroid pesticides, because there is a relatively reliable recovery for CHCs. This approach is less expensive due to reducing sample pre-treatment time and solvent consumption.

A screen-printed, amperometric biosensor for the determination of organophosphorus pesticides in water samples

An amperometric biosensor based on screen-printed electrodes （SPEs） was developed for the determination of organophosphorus pesticides in water samples. The extent of acetylcholinesterase （AChE） deactivation was determined and quantified for pesticide concentrations in water samples. An enzyme immobilization adsorption procedure and polyacrylamide gel matrix polymerization were used for fabrication of the biosensor, with minimal losses in enzyme activity. The optimal conditions for enzyme catalytic reaction on the SPEs surfaces were acetylthiocholine chloride （ATChCl） concentration of 5 mmol/L, pH 7 and reaction time of 4 min. The detection limits for three organophosphorus pesticides （dichlorvos, monocrotophs and parathion） were in the range of 4 to 7 μg/L when an AChE amount of 0.1 U was used for immobilization.

Enzyme Inhibition Rate Method for Rapid Detection of Organophosphorus and Carbamate Pesticides in Cowpea

[Objectives ] The paper was to explore enzyme inhibition rate method for rapid detection of organophosphorus and carbamate pesticides in cowpea. [ Methods ] Acetylcholinesterase （ACHE） was added to cowpea extract, to determine the inhibition rate of extract against enzyme. The influences of different sampiing methods and sampling parts on detection results were compared. [ Results] The positive rate of standard sampling was 18.18% higher than that of non-stand- ard sampling, and the positive rate of samples collected from cowpea tail was 16.67% higher than that collected from other parts. [ Condmions] Enzyme inhibi- tion rate method is suitable for rapid detection of organophosphorus and carbamate pesticides in cowpea.

Dynamic Microwave-assisted Extraction Online Coupled with QuEChERS for the Determination of Organophosphorus Pesticides in Cereals by Gas Chromatography

A rapid analytical method was first developed to detect organophosphorus pesticides(OPPs)in cereals,which used dynamic microwave assisted extraction(DMAE)coupled online with a modified quick,easy,cheap,effective,rugged,and safe(QuEChERS)method.Cereals samples were mixed with a certain amount of quartz,Ci8 and the primary second amine(PSA),and extracted successively with the acetonitrile-water solution(80%,VIV)under microwave irradiation.The obtained eluate was directly introduced into a collection tube containing NaCl and MgSO4.Finally,the supemate was evaporated and reconstructed,and determined by gas chromatographic spectrometry.Some factors affecting the experimental results were studied and optimized.The extraction and purification processes were carried out coinstantaneously and 12 samples could be treated in one step in 4 min.Low limits of tection(0.43—1.31 p.g/kg)for OPPs were obtained in cereals.The relative standard deviations(RSDs)were 2.1%—9.3%.The recoveries of OPPs ranged from 76.1%to 100.2%.By combining the advantages of DMAE with QuEChERS,the sample pretreatment process was simple to operate,with little amount of organic solvent.The whole extraction process was completed in a closed environment,therefore it was very appropriate for daily analysis of OPPs in cereals.

The Influencing Factors of Disposable Acetylcholinesterase Biosensor for in Situ Detection of Organophosphorus Pesticide

An amperometric biosensor based on acetylcholinesterase (ACHE) is assembled by simple adsorption of the AChE on 7,7,8,8-tetracyanoquinodimethane (TCNQ) modified screen-printed electrodes.This biosensor is used to detect the inhibitory effect of organophosphorus pesticides on AChE activity.The detection of organophosphorus pesticides is done with acetylthiocboline chloride (ATCh) as substrate.In order to obtain the optimized response to substrate,the influencing factors of the biosensor are investigated,including temperature,pH,incubation time,substrate concentration and AChE concentration. The measurements were performed after inhibition by immersing the enzyme electrode into the parathion methyl solution. Under the optimized conditions,that is,500 U/ml AChE concentration,pH 7.2,10 min incubation time,2 mmol/L substrate concentration and temperature of 37℃,from 5×10^(-8) mol/L to 5×10^(-5) mol/L is close to linear (regression equation: y(%)=124.055+15.7991gx,R^2=0.99644),which corresponds to 8.7%～56.1% AChE inhibition.With the optimized conditions, the lowest detectable amount of parathion methyl is 13×10^(-9).

Study on the toxic effect of the mixture of organophosphorus pesticide on perinatal rats

Objective To observe the toxic effect of the mixture of organophosphorus pesticide (MOP) on maternal rats and on the growth and development of their offspring. Methods Totally 40 Sprague-Dawley pregnant rats were randomly divided into three MOP dose groups and one control to which their offspring would be assigned. The experimental dosage of MOP and distilled water were administered orally starting on gestation day 15 and continued for 35 days. The physical development indices and the learning ability of F1 rats were measured during lactation. The pathological changes of uterus and liver of F0 rats were observed after weaning, while the weight ratio of uterus and some viscera to body of the F1 were examined. Results There were obvious changes of uterus and liver in the high-dose group of F0. The body-weight accretion of the F1 in high-dose group was obviously lower than that in control group (P<0.05). Some of the MOP F1 rats development indices delayed significantly (P<0.05), the learning ability decreased obviously, and the time of setting up memory prolonged (P<0.05). The ratio of the uterus weight to body-weight in the F1 MOP groups was significantly higher than that in control group (P<0.05). Conclusion The experiment doses of MOP are proved to have significant reproductive toxicity on perinatal rats.

Coordinatively unsaturated cobalt single-atom nanozymes for visual pesticides detection by smartphone-based platform

By adjusting the coordination environment of single-atom catalysts,the enzyme-like activity can be finely tuned for highly sensitive biosensing.Herein,we demonstrated that coordinatively unsaturated cobalt-nitrogen sites doped within porous carbon(SA-CoN_(3))could serve as highly efficient oxidase mimic.Compared with the typical planar four-coordination structure(SA-CoN_(4)),the as-obtained single-atom Co nanozymes anchored by three nitrogen atoms are found to display much higher oxidase-like catalytic efficiency.Combined theoretical and experimental analysis revealed that the coordinatively unsaturated Co sites could facilitate adsorption and activation of O_(2) molecule and thus improve their oxidase-like activity.Based on the enhanced oxidase-like activity of SA-CoN_(3),a paper/smartphone sensor for organophosphorus pesticides(OPs)was successfully constructed and used to quantify glyphosate in environmental and food samples with a low detection limit of 0.66μM.This work not only highlights the important role of coordination unsaturation of SA nanozymes for promoting oxidase-like activity,but also provides an easy and cost-effective way to conduct effective quantification of OPs in the field.

Pesticide Residue Content in Vegetable Bases of Lhasa City

[Objectives]To understand the quality safety of vegetables produced in vegetable planting bases around Lhasa City.[Methods]Four vegetable planting bases in the east,west,south,and north of Lhasa City were randomly selected for testing using rapid measuring instrument for agricultural residues and solid-phase microextraction-gas chromatography-mass spectrometer,with 120 vegetable samples selected.The testing samples included seven categories:green leafy vegetables,eggplants,melons,onions and garlic,root vegetables,beans,and fungi,totaling 29 types of vegetables.The samples were tested for 8 common prohibited and restricted pesticides in organic phosphorus and carbamate esters,including methamidophos,parathion,methyl parathion,monocrotophos,isocarbophos,methamidophos,aldicarb,and carbofuran.[Results]The pesticide inhibition rate of 6 kinds of vegetables detected by the rapid measuring instrument for agricultural residues was relatively high,with green leafy vegetables being the majority,reaching over 25%.By further establishing a solid-phase microextraction-gas chromatography-tandem mass spectrometry method,the accuracy of the data was verified experimentally.The experimental results indicated that the inhibition rate of vegetable samples in this experiment did not exceed 15%,and it was determined as a pollution-free vegetable.[Conclusions]According to the restriction standard of national pesticide use,the inhibition rate of vegetable samples was less than or equal to 40%,indicating that the quality of vegetables was qualified.

A dual-signal sensor for the analysis of parathion-methyl using silver nanoparticles modified with graphitic carbon nitride

A highly sensitive and selective method was developed for both UVevis spectrophotometric and fluorimetric determination of organophosphorus pesticides(OPs). This method used silver nanoparticles(AgNPs) modified with graphitic carbon nitride(g-C_3N_4). The AgNPs reduced the fluorescence intensity of g-C_3N_4. Acetylthiocholine(ATCh) could be catalytically hydrolyzed by acetylcholinesterase(AChE) to form thiocholine, which induces aggregation of the AgNPs. This aggregation led to the recovery of the blue fluorescence of g-C_3N_4, with excitation/emission peaks at 310/460 nm. This fluorescence intensity could be reduced again in the presence of OPs because of the inhibitory effect of OPs on the activity of AChE. The degree of reduction was found to be proportional to the concentration of OPs, and the limit of fluorometric detection was 0.0324 mg/L(S/N=3). In addition, the absorption of the g-C_3N_4/AgNPs at 390 nm decreased because of the aggregation of the AgNPs, but was recovered in presence of OPs because of the inhibition of enzyme activity by OPs. This method was successfully applied to the analysis of parathion-methyl in real samples.