enhanced competitive chemiluminescent enzyme immunoassay for the trace detection of insecticide...

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Enhanced Competitive Chemiluminescent EnzymeImmunoassay for the Trace Detection ofInsecticide TriazophosMaojun Jin, Hua Shao, Fen Jin, Wenjun Gui, Xiaomei Shi, Jing Wang, and Guonian Zhu

Abstract: A direct competitive chemiluminescent enzyme immunoassay (CLEIA) for triazophos was developed, whichwas based on the anti-THHe IgG monoclonal antibody and a heterogeneous enzyme tracer (THHu-HRP). Severalcomponents of chemiluminescent enhanced solution (CES) were optimized. The results showed that 1 mM of p-iodo-phenol, 0.625 mM of luminol, and 4 mM of H2O2 had the best performance. Based on the study of CES, the influenceof several factors (assay buffer, blocking substance, and solvent) on the immunoassay was investigated. The sensitivity fordetection, IC50 value was 0.87 ng/mL at a practical working concentration range between 0.04 ng/mL and 5 ng/mLand the limit of detection for triazophos was 0.063 ng/mL. The average recovery of triazophos added to lettuce, carrot,apple, water, and soil were 78.71%, 67.52%, 118.3%, 117.2%, and 122.0%, respectively. Finally, comparison betweenthe methods of CLEIA and high-performance liquid chromatography-tandem mass spectrum (HPLC-MS/MS) wasperformed. The results obtained from CLEIA were in agreement with those of HPLC-MS/MS.

Keywords: chemiluminescent enzyme immunoassay, enhancement, high-performance liquid chromatography-tandemmass spectrum, triazophos

Practical Application: The method developed in the paper could take positive or negative test of vegetable, fruit, andenvironmental samples for triazophos with high sensitivity and efficiency.

IntroductionTriazophos [O,O-diethyl O-1-phenyl-1H-1,2,4-triazol-3-yl

phosphorothioate] is a broad-spectrum organophosphate insec-ticide and acarcide with nematicidal properties. In recent years,most high-toxic and high-residual organophosphorus pesticides,for example, parathion, methyl parathion, and methamidophoswere banned in crops by Chinese Ministry of Agriculture. As agood alternative to the above pesticides, triazophos is widely usedon a variety of crops and now is one of the most important pes-ticides for controlling bollworm on paddy fields in China (Liu1999). Triazophos is moderately toxic to mammals, but highlytoxic to fish and honeybees. Due to its residue in a large num-ber of foods, potential health hazards of triazophos residue havebeen recognized by the scientists (Holden and others 2001; Li andothers 2004).

The gas chromatography (Gong and others 2004; Zhang andothers 2004; Li and others 2008) and high-performance liquidchromatography (HPLC) (Fu and others 2009) have been estab-lished for the analysis of triazophos. However, these methods havesome disadvantages that complicated concentration and derivati-zation steps are needed to obtain the desired sensitivity, and the

MS 20110404 Submitted 3/31/2011, Accepted 1/9/2012. Authors M. Jin, Shao,F. Jin, Shi, and Wang are with Key Laboratory for Agro-Products Quality and Safety,Inst. of Quality Standards, Testing Technology for Agro-Products, Chinese Academy ofAgricultural Sciences, Beijing, 100081, China. Author Gui is with Inst. of Pesticideand Environmental Toxicology, Zhejiang Univ., Hangzhou, 310029, China. Directinquiries to author Wang (E-mail: [email protected]).

heavy use of toxic solvents is harmful to operators. Furthermore,these methods require skilled analysts, expensive instruments, andthey are not very suitable for high-throughput detection. Im-munoassays are demonstrated as simple, rapid, and cost-effectivealternatives to the traditional methods. Recently, photothermalflow injection analysis (FIA) system based on cholinesterase in-hibition, enzyme-linked immunosorbent assay (ELISA), and im-munochromatographic assay based on use of monoclonal antibody(MAb) was successfully developed for the detection of triazophosresidues (Pogacnik and Franko 2001; Gui and others 2006; Liangand others 2007; Gui and others 2008).

Chemiluminescence (CL) is the production of electromagneticradiation by a chemical reaction. The CL intensity is directly pro-portional to the concentration of a limiting reactant involved inthe CL reaction. CL has been exploited for its use in a widerange of applications due to its high sensitivity, wide calibrationrange, and suitable for miniaturization (Fan and others 2009; Jungand others 2011). Therefore it plays a significant role in medi-cal testing and environmental monitoring nowadays (Hayama andothers 2010; Liu and others 2010; Vdovenko and others 2010;Xin and others 2010). However, the applications of chemilumi-nescent immunoassay in detection of pesticide residues were rarelyreported. The horseradish peroxidase (HRP)-catalyzed chemilu-minescent oxidation of luminol is widely used in chemilumines-cent immunoassay. Moreover, the enhancer used in the assay hasthe ability to produce intense and stable light emission for a longperiod of time, which makes it compatible with microplate assayformats. In this paper, chemiluminescent enzyme immunoassayhas been applied to the determination of triazophos, and a rapid

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Immunoassay for triazophos . . .

and highly sensitive screening method was developed for moni-toring triazophos residues in vegetables.

Experimental

Immunoreagents, materials, and instrumentsThe synthesis of haptens (THHe and THHu) and the prepa-

ration of MAb have been described in the previous paper (Guiand others 2006). THHu-HRP was prepared using the mixed-anhydride method (Rajkowski and others 1977). Triazophos stan-dard was obtained from Hangzhou Geling Scientific Instrument(Hangzhou, China). The standard was dissolved in methanol toprepare a 100 ng/mL stock solution and stored at –20 ◦C untilused. Triazophos is hazardous and should be handled with care inexperiment. Bovine serum albumin, ovalbumin, Tween 20, HRP,p-iodo-phenol (PIP), and Trometamol (Tris) were purchased fromSigma-Aldrich (St. Louis, Mo., U.S.A.). Luminol was supplied byFluka Tryptone. All other chemicals and organic solvents were ofanalytical grade or better.

The white opaque 96-flat-bottomed well plates were purchasedfrom Corning (COSTAR, Lowell, Mass., U.S.A.). Plates werewashed with a DEM plate washer (Beijing Tuopu Analytical In-struments Co. Ltd., China), and chemiluminescent intensity wasmeasured with a chemiluminescent detector (Promega, Madison,Wis., U.S.A.).

Chromatographic analyses were conducted using an Agilent1200 HPLC system (Agilent, Santa Clara, Calif., U.S.A.) equippedwith a binary pump, a column oven, and an auto sampler. Massspectrometric detection was carried out using an API 5000 tan-dem quadrupole mass spectrometer (Applied Biosystems, FosterCity, Calif., U.S.A.) in the multiple-reaction monitoring (MRM)mode. The instrument was equipped with an electrospray ioniza-tion source (ESI). All system control, data acquisition, and dataanalysis were performed with the AB Sciex Analyst 1.4.2 software(Applied Biosystems, Foster City, Calif., U.S.A.).

CLEIA protocolThe immunoassay to be optimized was a direct CLEIA based

on the coating anti-THHe MAb and the heterogeneous enzymetracer (THHu-HRP). All incubations were carried out at 37 ◦C.Standards were prepared in Tris-HCl buffer (80 mM Tris, pH 7.2)containing 5% methanol by serial dilutions from a stock solutionin methanol. The white opaque 96-flat-bottomed well plates werecoated with anti-THHe MAb in 80 mM Tris-HCl buffer (pH 7.2)and incubated at 37 ◦C for 2 h. After the plate was washed 3 timeswith PBST (PBS containing 0.05% Tween 20, pH 7.4), free bind-ing sites of the wells were blocked with 300 μL/well of 1% skimmilk in 80 mM Tris-HCl buffer (pH 7.2) for 30 min at 37 ◦C.Afterward, 50 μL/well of triazophos standard or pretreated sam-ples were added, followed by adding 50 μL/well of THHu-HRPin 80 mM Tris-HCl buffer (pH 7.2) containing 2% skim milk.After incubation at 37 ◦C for 1 h and 3 more extensive washes,200 μL/well of chemiluminescent enhanced solution (CES) wasadded to the plates. The chemiluminometric signal generated fromthe HRP-luminol-H2O2 system was measured at 425 nm withthe Promega chemiluminescent detector. The luminous intensityis represented as relative light units (RLU).

Sample preparationBlank samples of lettuce, carrot, apple, and pear were purchased

from a local supermarket in Beijing. For sample homogenization,1 kg of lettuce, carrot, apple, and pear were chopped into small

pieces thoroughly and then transferred to a homogenizer (PhilipsElectronics Ltd., Holland) and ground to homogeneous matrices.

For CLEIA method, 10 g of the samples were weighed andspiked with the trizaophos standard in acetonitrile, followed byextraction with acetonitrile by vigorously shaking with hand for1 min. Then, 1 g of sodium chloride and 4 g of magnesiumsulphate were added to the samples. With another 1 min of strongshaking by hand, the solutions were centrifuged at 3000 rpm for5 min at room temperature. Afterward, 1 mL of the upper layerwas transferred to glass tube and evaporated at 40 ◦C under gentlenitrogen stream. Subsequently, the dry residues were diluted withthe mix solution of Tris-HCl buffer and methanol (19/1, v/v)prior to CLEIA.

For HPLC-MS/MS method, the extraction and cleanup pro-cedures were similar to the procedures of CLEIA. The proceduresfor extraction and cleanup were described below: (1) 10 g of carrotsamples were weighed and spiked with the triozophos standard at5 ng/g, 10 ng/g, 15 ng/g, and 20 ng/g; (2) 10 mL of acetonitrilewere added to the samples and the samples were shaken vigorouslyby hand for 1 min; (3) 1 g of sodium chloride and 4 g of mag-nesium sulphate were added to the samples and shaked vigorouslyfor another 1 min; (4) after centrifuge (3000 rpm, 5 min), 4 mL ofupper layer was transferred to a plastic tube; (5) 0.6 g of magnesiumsulphate and 0.2 g of primary secondary amine (PSA) were addedand shaken by hand for 30 s; (6) after standing for 1 min, 1.5 mL ofsupernatant fluid was filtered with 0.22 μm organic microfiltrationmembrane prior to HPLC-MS/MS (Lehotay 2005).

Optimization of chemiluminescent enhanced solutionWith the aim to get an excellent CES, the influence of sev-

eral components of CES was examined systematically. The effectof different chemiluminescent enhancers including bromophenol,PIP, sodium tetraphenyl borate, trans-4-hydroxycinnamic acid wasinvestigated at first. After selecting the best chemiluminescent en-hancer, the influence of its concentration on CL intensity wasstudied. And then, the effect of luminol at a serial of concentra-tion was evaluated. Finally, the influence of oxidant at differentconcentration was studied.

HPLC-MS/MS analysisTriazohpos was separated on an XTerra C18 (150 mm ×

2.1 mm, 3.5 μm) column (Waters, Milford, Mass., U.S.A.). Themobile phases consisted of phase A (0.1% formic acid in purewater) and B of acetonitrile was used with a gradient elution ofA: B = 85: 15 to 5:95 (0 to 0.5 min, hold for 3.5 min), 85:15(4.0 to 4.5 min, hold for 5.5 min) at a flow rate of 0.2 mL/min.The injection volume was 5 μL, and the column temperature wasmaintained at 40 ◦C.

The MS conditions for the determination of triazophos was firstinvestigated using FIA. The triazophos standard at 50 μg/L wasused to obtain the optimum MS/MS parameters. Mass spectrumrange was from m/z 50 to 350 amu. The precursor ion [M+H]+at m/z of 314.3 corresponding to triazophos was found in the fullscan spectrum. The typical product ions at m/z of 115.0 and 162.2were monitored, and m/z 115.0 was used for quantification. TheMRM mode was used, and typical parameters were used as follows:Ion spray voltage (IS): 4500 V; Atomization air pressure (GS1):50 Psi; Auxiliary gas (GS2): 30 Psi; Curtain gas (CUR): 10 Psi; Ionsource temperature (TEM): 350 ◦C; Entrance potential (EP): 10 V;Collision cell exit potential (CXP): 13 V. The MRM transitionsas well as collision energy (CE) and declustering potential (DP)applied were summarized in Table 1.

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Results and Discussions

Chemiluminescent enhanced solutionBromophenol, PIP, sodium tetraphenyl borate, trans-4-

Hydroxycinnamic acid were selected as the chemiluminescentenhancer, and the difference of the enhancement was investi-gated. The concentrations of 4 enhancers were varied from 0.05mM to 2 mM, and the concentrations of luminol and H2O2

were fixed at 1 mM and 5 mM, respectively. The maximum en-hanced chemiluminescent intensities obtained for bromophenol,PIP, sodium tetraphenyl borate and trans-4-Hydroxycinnamic acidwere 8448943, 16402033, 28720, and 7092280. The CL intensi-ties in the presence of various concentrations of PIP were given inFigure 1. The result showed that when the concentration of PIPwas 1 mM, the maximum RLU was obtained.

Subsequently, the concentrations of luminol were optimized.At fixed concentrations of other components in CES, a series ofconcentrations of luminal ranging from 0.04 mM to15 mM werestudied. The results were shown in Figure 2, and it showed thatmaximum RLU was achieved at 0.625 mM of luminol.

H2O2 was selected as the oxidant in the system, and the con-centrations in the range of 0.25 to 48 mM were investigated. Theresults were shown in Figure 3, and it showed that 4 mM of H2O2

gave the maximum RLU.Taking all the factors into account, the optimized conditions for

the CES were summarized as follows: 1 mM of PIP, 0.625 mM of

Table 1–Molecular weights, retention times, and optimized MS/MSparameters for triazophos.

ESI MRM DP CE EP CXPmode M.W. RT transition (V) (V) (V) (V)

Positive 313.3 5.12 ± 0.2 313.2→115.0 90 40 10 13313.2→162.2 90 28 10 13

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Figure 1–Effect of the concentration of PIP on the RLU of immunoassay.Each point represents the average of 4 replicates.

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Figure 2–Effect of the concentration of luminol on the RLU of immunoassay.Each point represents the average of 4 replicates.

luminol, and 4 mM of H2O2 were dissolved in 100 mM Tis-HClbuffer (pH 8.5).

Development of CLEIASome studies (Abad and others 1998; Casino and others 2001;

Lee and others 2001) revealed that salt concentration and pH hada profound effect on both signal and sensitivity. Several buffers fre-quently used in immunoassay were optimized to choose a best onefor the method. The results showed that the buffer for maximumRLU and the buffer for lowest IC50 were different. Therefore theratio of maximum RLU and IC50 value was used as the criteriato evaluate the performance. The results were given in Table 2.From the results, it showed that 80 mM Tris-HCl buffer (pH 7.2)was most suitable to the immunoassay.

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Figure 3–Effect of the concentration of H2O2 on the RLU of immunoassay.Each point represents the average of 4 replicates.

Table 2–Comparison of assay buffers on the immunoassay.

Salt concentrationBuffer pH (mol/L) IC50(ng/mL) RLUmax

Tris-HCl 7.2 0.02 0.919 284719450.05 0.872 267856570.08 0.831 251984610.10 0.865 24698913

PBS 7.4 0.01 0.887 251894150.02 0.852 265401920.05 0.927 231065740.08 1.060 207649140.10 1.215 18610651

Note: In the experiment, 2% skim milk was chosen as blocking substance. Anti-THHeMAb: 10 mg/L, THHu-HRP: 0.2 mg/L were ascertained as working concentration.

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Figure 4–Effect of the concentration of methanol on the analytical charac-teristics of triazophos standard curve: (�) RLU in the absence of triazophos(RLUmax) and ( ) value of IC50 for triazophos. Each point represents theaverage of 4 replicates.

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Being a less polar pesticide, triazophos was usually dissolved inorganic solvent to make a stock solution and the working solu-tions were prepared with serial dilutions in buffer prior to theimmunoassay. According to many studies, methanol caused theleast negative effect to the immunoassay (Manclus and Montoya1996). Therefore, it was necessary to investigate the effect of var-ious amount of methanol in buffer on immunoassays. The resultwas given in Figure 4. From the result, it might be speculated thatincreasing the content of methanol caused a decrease in RLU andan increase in IC50. When the concentration of the methanol inthe buffer was 5%, the highest ratio of maximum RLU to IC50

value was obtained.

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Figure 5–Effect of the concentration of skim milk as blocking substance onthe analytical characteristics of triazophos standard curve: (�) RLU in theabsence of triazophos (RLUmax) and ( ) value of IC50 for triazophos. Eachpoint represents the average of 4 replicates.

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Figure 6–Typical standard curves for triazophos by CLEIA under optimizedconditions. Data represent the means of 4 determinations.

Blocking substance was used in the immunoassay to reducenonspecific binding or improve sensitivity, and skim milk wasthe common blocking substance in previous paper. In our work,various concentrations of skim milk as blocking solution wereexamined. The result was given in Figure 5. The results showedthat 1% of skim milk was the best choice in the experiment.

Taking all these factors into account, the optimized conditionsfor the immunoassay were summarized as follows: a coating MAb(anti-THHe IgG) concentration of 10 mg/L, an enzyme tracer(THHu-HRP) concentration of 100 μg/L, 37 ◦C for all incu-bations, 2 h incubation for coating step, 30 min incubation forblocking step in buffer containing 1% skim milk, an incubationtime of 1 h for the competitive step, a 5 min for the reactionbetween CES and HRP, 80 mM Tris-HCl buffer (pH 7.2) with1% skim milk for the competitive step and 5% methanol in assaybuffer.

Determination of dynamic range and limit of detectionThe standard curve for triazophos was established in the con-

centration range of 0 to 20 ng/g based on the average of 4 replicatemeasurements for each concentration. The results were shown inFigure 6. A linear correlation between the inhibition rate and tri-azophos concentration was obtained between 0.039 and 5 ng/mL.The regression equation was y = 15.007Ln(x) + 52.026. The IC50

for triazophos was 0.87 ng/mL, and IC10 was nearly 0.06 ng/mL.The range of coefficient of variation was approximately 2.18% to13.67%. The limits of detection (LOD) and quantification (LOQ)of triazophos of CLEIA were estimated by analyzing the blanksolution. LOD and LOQ were calculated based on a standard de-viation (SD) value of the 10 replicate determinations. The detailedcalculation is as follows: the inhibition rate (3 SD/RLU0 × 100%,10 SD/RLU0 × 100%) were got at first. Then the inhibition ratevalues were put in the regression equation to obtain the corre-sponding concentrations to the values of 3 SD/RLU0 × 100%and 10 SD/RLU0 × 100%, which were the values of LOD andLOQ (RLU0 means the RLU value of blank solution). Based onthe calculation, the LOD and LOQ of triazophos of CLEIA were0.063 ng/mL and 0.327 ng/mL.

Spiked recoveryIn order to evaluate the feasibility of applying the proposed im-

munoassay to real samples, triazophos was spiked in water, soil, andvegetables. To correct for the matrix effect, blank samples wereinitially extracted in mixed solution (80 mM, pH 7.2 Tris-HClsolution/methanol = 19/1, v/v, the same below) and analyzedwith the CLEIA method. The water and soil samples were spikedwith triazophos at 1 and 5 ng/g and the lettuce, carrot, and apple

Table 3–Reproducibility and recovery of triazophos from spiked samples by CLEIA.

Mean background value Spiked aMean ± SD Mean recoverySamples Dilutions (ng/g, n = 8) level (ng/g) (ng/g) (%, n = 8) C.V. (%)

Lettuce 4 1.636 10.0 10.32 ± 2.061 86.81 19.9720.0 15.76 ± 2.733 70.61 17.34

Carrot 4 1.464 10.0 8.040 ± 1.632 65.76 20.3020.0 15.32 ± 2.589 69.28 16.90

Apple 4 3.372 10.0 15.64 ± 3.372 122.7 21.5620.0 26.16 ± 5.352 113.9 20.49

Water 2 0.322 1.00 1.536 ± 0.275 121.4 17.905.00 5.972 ± 1.212 113.0 20.29

Soil 2 0.524 1.00 1.824 ± 0.418 130.0 22.925.00 6.228 ± 1.236 114.1 19.84

aThe mean determination value included the background value.


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samples were spiked at 10 and 20 ng/g, as described in “Samplepreparation” section. Subsequently, the pretreated samples abovewere determined by the CLEIA method directly. The resultswere given in Table 3. Precision obtained for all samples meetthe demand for a residue method, since most coefficients of vari-ation were around or below 20%, and so were the recoveries (Themean was 78.71%, 67.52%, 118.3%, 117.2%, and 122.0% for let-tuce, carrot, apple, water, and soil, respectively). The nonspikedsamples were also included in the analysis as negative controls.

Despite the improvement in sensitivity, the sample matricescaused severe interferences with the assay. Take apple as an exam-ple, the reaction between the antibody and hapten was inhibited37.6% in the undiluted sample. As a result, suitable dilution wasnecessary to decrease the matrix interference.

Confirmation testIn the study of HPLC-MS/MS method validation, calibration

curve was prepared using matrix-matched standard samples, widelinear ranges was 1 to 100 μg/L for triazophos, correlation coef-ficients (r2) was 0.9983. As shown in Table 4, satisfactory methodrecoveries was obtained for the triazophos spiked at 3 concen-tration levels in carrot sample (88.16% to 97.4%, RSD <9%).

Table 4–Reproducibility and recovery of triazophos from carrot byHPLC-MS/MS.

Spiked Meanlevel recovery

Sample (ng/g) (%, n = 4) C.V. (%) LOD (ng/g) LOQ (ng/g)

Carrot 5.0 88.16 8.13 0.17 0.5610.0 97.40 5.2120.0 95.63 3.19

LOD and LOQ of triazophos of HPLC-MS/MS were estimatedby analyzing matrix-matched standard at 10 ng/g. LOD and LOQwere calculated based on a peak to peak signal-to-noise value, thatwas S/N = 3 and S/N = 10, respectively. The typical HPLC-MS/MS chromatograms for the samples used as a carrot matrixblank sample, matrix-matched standard at 10 ng/g, and a carrotsample spiked at 10 ng/g were showed in Figure 7.

The CLEIA developed in this work was compared with theHPLC-MS/MS in the determination of triazophos. Thirty-sixcarrot samples were spiked at 5, 10, and 15 ng/g. Half of the

Table 5–Analysis by CLEIA and HPLC-MS/MS of carrot spiked withtriazophos at 5, 10, and 15 ng/g.

Spiked Detection of Detection ofconcentration HPLC-MS/MS method CLEIA method

Nr (ng/g) (ng/g) (ng/g)

1 5.0 4.21 4.892 5.0 4.48 3.733 5.0 4.72 3.174 5.0 4.93 3.145 5.0 4.06 3.076 5.0 4.17 3.307 10.0 9.89 6.878 10.0 9.04 8.659 10.0 9.77 6.74

10 10.0 10.21 7.4811 10.0 9.80 7.8712 10.0 9.43 6.2913 15.0 13.59 8.1714 15.0 13.78 11.2215 15.0 14.13 9.5016 15.0 13.33 10.3817 15.0 13.15 9.7918 15.0 14.46 11.14

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Figure 7–The typical HPLC-MS/MSchromatograms for the samples used as a carrotmatrix blank sample (A), matrix-matchedstandard at 10 ng/g (B), and a carrot samplespiked at 10 ng/g (C).

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y = 1.3034x + 0.2055

R2 = 0.8996

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Figure 8–Correlation between concentrations of triazophos measured byCLEIA and by HPLC-MS/MS in carrot samples.

samples were analyzed by HPLC-MS/MS and the other half byCLEIA. The results were given in Table 5 and Figure 8. A linearcorrelation was observed between these 2 methods for the deter-minations of triazophos in carrot samples. The regression equationwas y = 1.303x + 0.205, with R2 value of 0.8996.

The validation outcome showed that each assay was robust,reliable, and accurate to meet the requirements of the intendedanalytical application, namely: (1) semiquantitative determinationof the concentration of triazophos in the vegetable, fruit, andenvironmental samples and (2) positive or negative test of a samplefor triazophos. Of course, the greatest value of the CLEIA methodis to provide a choice for positive screening of the samples withhigh sensitivity.

ConclusionsWith the enhanced chemiluminescence of the HRP-luminol-

H2O2 system, a high-throughout and sensitive CLIA method wasdeveloped for the determination of triazophos in vegetable, fruit,and environmental samples. The detection limit for triazophos was0.06 ng/mL and the limit of quantification was below 10 ng/mL,which was lower than the MRL value of triazophos set by CACand any other countries. Meanwhile, the proposed method showedgood spike recovery of approximately 66% to 130% and goodcorrelation with the results of HPLC-MS/MS, indicating that itcould meet the demands in daily positive screening for a largenumber of samples.

AcknowledgmentsThis study was supported by National Science & Technology

Cooperation Project of Chinese Ministry of Science & Technol-ogy (S2012ZR02020), Chinese Public-interest Industrial Science& Technology Project (201203094), World Bank Loan Project in

Jilin Province Agro-product Quality and Safety Project (2011-Z5),and Fundamental Research Funds for Chinese Central Public-interest Institution (0032011011). The authors state that there areno conflicts of interest.

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enhanced competitive chemiluminescent enzyme immunoassay for the trace detection of insecticide...

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