A Recombinant Fluorescent Peptidomimetic Tracer for

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A Recombinant Fluorescent Peptidomimetic Tracer for Immunodetection of Imidaclothiz Yuan Ding, Xiude Hua, Mei Du, Qian Yang, Lina Hou, Limin Wang, Fengquan Liu, Gualberto G. Gonzalez-Sapienza, and MingHua Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03685 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 5, 2018

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Analytical Chemistry

A Recombinant Fluorescent Peptidomimetic Tracer for Immunodetection of Imidaclothiz Yuan Ding †, ‡, Xiude Hua*, †, ‡, Mei Du †, ‡, Qian Yang †, ‡, Lina Hou †, ‡, Limin Wang †, ‡, Fengquan Liu †, ‡, §,

Gualberto Gonzalez-Sapienza⊥, Minghua Wang†, ‡

†College

‡

of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

State & Local Joint Engineering Research Center of Green Pesticide Invention and Application,

Nanjing 210095, China §Institute

of Plant Protection, Jiangsu Academy of Agricultural Science, Nanjing 210014, China

⊥Cátedra

de Inmunología, Facultad de Química, Instituto de Higiene, Universidad de la República,

Montevideo 11600, Uruguay * Tel.: +86 25 84395479. Fax: +86 25 84395479. E-mail address: [email protected].

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ABSTRACT Peptidomimetic and anti-immunocomplex peptides, which could be readily isolated from phage display library, have shown a great potential for small molecule immunoassay development because they typically improve the sensitivity and avoid the use of chemical haptens as coating or tracers antigens. However, the phage borne peptides are unconventional immunoassay reagent, which greatly limits their use in commercial applications and requires secondary reagents for detection. In order to overcome these limitations, we used C2-15, a peptidomimetic of imidaclothiz, as a model peptide fused to emerald green fluorescent protein (EmGFP) at the N terminus (C2-15-EmGFP) and C terminus (EmGFP-C2-15) to generate novel fluorescent peptide tracers. Both recombinant fluorophores reacted with similar affinity to the anti-imidaclothiz monoclonal antibody 1E7, but due to its higher expression C2-15-EmGFP was chosen to develop a competitive magnetic separation fluorescence

immunoassay

(MSFIA).

After

a

competitive

step

with

the

analyte,

the

C2-15-EmGFP/antibody complex bound to the magnetic beads was separated with a magnet, and due to the fast dissociation of the peptide-antibody interaction, the fluorescence signal was detected following the spontaneous dissociation of the complex in fresh buffer. The concentration of imidaclothiz causing the 50% inhibitory concentration (IC50) was 11.00 ng mL-1, and the MSFIA performed

with

excellent

recovery

and

good

correlation

with

high-performance

liquid

chromatography in different matrices. Keywords: Peptidomimetic; Recombinant peptide; Emerald green fluorescent protein; Magnetic nanoparticles; Imidaclothiz


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INTRODUCTION Phage display peptide technology has been successfully used to provide peptide reagents for immunodetection of small molecules by using two formats: a) by serving as peptidomimetics of the analyte, which react with the anti-analyte antibody to develop competitive immunoassays,1-4 and b) by serving as anti-immunocomplex specific binders, which react with the analyte-antibody immunocomplex enabling the development of noncompetitive immunoassays.3, 5-7 These peptides can be isolated from phage display libraries in a simple and cost-effective way and can be directly used as phage borne peptides for immunoassay development.8 However, the filamentous structure of the phage particle can be troublesome for the development of some immunoassay applications, such as immunochromatography tests. In addition, its detection requires secondary reagents, and their biological safety hazard presents some limitations to its use, particularly in commercial applications. Different strategies have been devised for overcoming these limitations using the peptides isolated by phage display in phage free immunoassays. In some reports, the synthetic versions of these peptides have been conjugated to either signal generating molecules or carrier protein for sensitive detection of small analytes.9-12 Alternatively, the encoding sequences of the peptides have been used to generate recombinant chimeric tracers or coating antigens, which reduces costs and avoids the hassles and batch-to-batch variation related to the chemical conjugation.12-14 In this work we explored the use of a fluorescent protein to substitute for the phage scaffold to produce a chimeric tracer that would not require labeling steps, secondary reagents for detection, or substrates. To the best of our knowledge, there are only two reports using peptidomimetics-based recombinant tracers. González-Techera et al developed a chimeric tracer by fusing a peptidomimetic



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of the herbicide molinate to the alkaline phosphatase of E. coli, but despite using a mutated form of the phosphatase the assay required hours to generate a measurable signal.13 More recently, our lab generated a fusion protein expressing a peptidomimetic of imidaclothiz at the N- and C-terminus of the deep sea shrimp luciferase (nano luciferase) and used the recombinant chimeras to developed the bioluminescent immunoassays.15 The recombinant chimera showed similar specificity and even better sensitivity than the phage enzyme linked immunosorbent assay (ELISA), but as in all enzymatic assays, it was necessary to include a step to add the luciferase substrate.16 In order to develop a faster and simpler assay, in this work we substitute the luciferase by a fluorescent protein that could be measured directly, using the Emerald green fluorescent protein (EmGFP). Since its introduction as recombinant protein in the nineties, the green fluorescent protein (GFP) from the jellyfish Aequoreavictoria has been widely used in biological and biotechnological applications. It has a stable fluorescence signal and strong photobleaching resistance. EmGFP, which is a protein engineered variant of GFP with improved photostability and brightness, was selected for this study as the protein partner to be fused to the peptidomimetic of imidaclotiz.17 The fluorescent peptide tracer was used to develop a magnetic separation fluorescence immunoassay (MSFIA) for immunodetection of imidaclothiz, employing functionalized magnetic nanoparticles (MNPs) as reaction and separation platform. MATERIALS AND METHODS Reagents. The monoclonal antibody (mAb) 1E7 and the cyclic 8-amino-acid peptidomimetic (C2-15, CLPPRMIYEC) were prepared as described previously.16,

18

The anti-His tag and anti-EmGFP tag

antibodies were purchased from Abcam (Cambridge, UK). The pRSET/EmGFP vector and pET-22b(+)


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vector were purchased from Invitrogen (California, USA) and Novagen (Darmstadt, GER), respectively. The HisTrap HP column for purification of recombinant proteins was purchased from GE Healthcare (Piscataway, NJ). The BCA Protein Assay Kit for quantification of recombinant proteins was provided by Pierce (Rockford, IL). The reagents for Fe3O4 MNPs synthesis and labeling were from Sigma (St. Louis, MO). Assembling of the Fluorescent Peptide Tracer Genes into the pET-22b(+) Vector. The EmGFP gene was amplified from the pRSET/EmGFP vector. The genes for expression of the peptide/EmGFP chimeras were assembled using the FN forward and RN reverse primers for C2-15-EmGFP (the peptide at the N-terminus), or the FC forward and RC reverse primers for EmGFP-C2-15 (the peptide at the C-terminus), respectively (Figure 1). After digestion and ligation, the chimeric gene fragments were inserted into the pET-22b(+) vector between the 6×His tag and the signal peptide pelB. The resulting vector was transformed into E. coli JM109 competent cells for amplification and DNA sequencing validation. After sequence confirmation, it was transformed into E. coli BL21(DE3) competent cell. Preparation of the Fluorescent Peptide Tracers. The E. coli BL21(DE3) cells containing the expression vector were cultivated in 2×YT culture medium with 50 μg mL-1 carbenicillin at 37 °C and 250 rpm. When the OD600 of the culture medium reached 0.8 AU, protein expression was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside, and the cells were further cultured at 20 °C and 250 rpm for 12 h. The cells were then collected by centrifugation and suspended in 20% sucrose solution (30 mM Tris-HCl, pH=8.0) with 1 mM ethylenediaminetetraacetic acid disodium salt. After stirring for 10 min, the cells were centrifuged and resuspended in cold 5 mM MgSO4 to release the periplasmic proteins. The purification procedure was carried on an ÄKTA avant 25 employing a 1 mL



HisTrap HP column. After removing the imidazol by ultrafiltration, the concentration of the recombinant protein was determined by the BCA Protein Assay Kit (Pierce, Illinois). The recombinant protein solution was supplemented with complete protease inhibitor and stored at -80 °C until used. Competitive ELISA Using the Fluorescent Peptide Tracers. The recombinant tracers were coated on clear microplate at 100 μg mL-1 (100 μL per well) in PBS. After washing 5 times with PBS containing 0.05% Tween 20 (PBST), the plate was blocked with 5% skimmed milk in PBS for 2 h at 37 °C. After washing, 50 μL mAb 1E7 (100 μg mL-1) was mixed with an equal volume of the imidaclothiz standard and this solution was added to the plate wells. After 1 h incubation at 37 °C, the plate was washed and 100 μL of horseradish peroxidase (HRP) labeled anti-mouse IgG antibody (1:20000 dilution in PBS) was added to the wells. After 1 h incubation and washing, 100 μL of HRP substrate was added to the wells, and the reaction was stopped by addition of 2 M H2SO4 (50 μL per well) after 15 min. The absorbance at 450 nm was measured in a Spectra-Max M5 reader (Molecular Devices). Preparation of Antibody-Labeled Fe3O4 MNPs. The synthesis and label of Fe3O4 MNPs were based on a previous study.19 Briefly, 1.0 g FeCl3·6H2O, 6.5 g 1,6-hexamethylenediamine and 2.0 g anhydrous sodium acetate were dissolved in 30 mL ethylene glycol at 50 °C. The mixture was heated at 196 °C and reacted for 6 h to form amino-functionalized Fe3O4 MNPs, which were then washed with ultrapure water and ethanol. The mAb 1E7 was conjugated to the MNPs by cross-linking with glutaraldehyde. Ten milligrams of Fe3O4 MNPs were dispersed in PBS and supplemented with 100 mg sodium borohydride and 1.25 mL 25% glutaraldehyde solution. After reacting for 1 h under gentle shaking, the unreacted reagents were removed by magnetic separations and the MNPs were reacted


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with 1 mL of mAb 1E7 (1 mg mL-1) in PBS for 6 h. Then 5 mL 1% BSA were added to block the remaining aldehyde groups for another 6 h. The mAb-labeled MNPs were separated by using a magnetic separator, washed and dispersed in 5 mL of PBS. MSFIA Assay. The scheme of the MSFIA is shown in Figure 2. One hundred microliters of C2-15-EmGFP (100 μg mL-1) in PBS were mixed in an Eppendorf tube with 200 μL of the imidaclothiz standard solution or sample solution (containing 5% methanol in PBS). Then 100 μL of mAb-labeled MNPs were added and incubated for 1 h at 25 °C under gentle rocking. The MNPs were separated by using a magnet and washed 3 times with PBS in 5 min. Washing time was always the same to avoid variations in the amount of tracers that dissociates during this step. The MNPs were then redispersed into 300 μL of PBS and gently rocked for 25 min at 25 °C. The full volume of the final MNPs suspension was transferred to white opaque microtiter plates to measure the fluorescence intensity by Spectra-Max M5 reader. The excitation/emission wavelengths were set at 485 nm/510 nm. Cross-Reactivity (CR). The reactivity of other neonicotinoid compounds was tested with the imidaclothiz MSFIA, and their CRs were calculated based on the values of the 50% inhibitory concentration (IC50) using the following formula: CR = [IC50 (imidaclothiz) / IC50 (analogue)] × 100%. Analysis of Spiked Samples. Blank samples of paddy water, soil, cucumber and brown rice were obtained from farms in Nanjing, China, and were verified by HPLC to ensure they were imidaclothiz-free. These samples were homogenized and spiked with imidaclothiz (4, 20, 100 ng mL-1 for paddy water, 40, 200, 1000 ng g-1 for soil and cucumber, and 80, 400, 2000 ng g-1 for brown rice).



The paddy water samples were directly analyzed by MSFIA after mixing with an equal volume of 2× PBS buffer. The solid samples (10 g) were extracted with 20 mL of 25% methanol-PBS, the mixture was intensely vortexed for 5 min and was sonicated for 15 min. After vacuum filtering, the solutions were collected and adjusted to 25 mL with PBS, and appropriate dilutions in PBS were analyzed by MSFIA. Each spiked sample was performed in triplicate. Correlation with HPLC. MSFIA and HPLC were used to analyze imidaclothiz in cucumber samples that were collected from local farms in Nanjing, China. Samples for MSFIA were prepared as above. For HPLC analysis, 40 mL acetonitrile were added to 10 g of cucumber to extract the pesticide by high-speed dispersion for 5 min followed by sonication for 15 min. After centrifugation, 5 g NaCl was added into the supernatants to separate the organic phase. Twenty milliliters of organic phase was collected by filtering through anhydrous sodium sulfate to remove the residual moisture. The extract was evaporated to dryness and dissolved in 2 mL of water:methanol = 60:40 (v/v). The sample solutions were injected into HPLC (Agilent 1260) with an Eclipse pluse-C18 column (250 mm × 4.6 mm, 5μm) to detect the concentration of imidaclothiz. The parameters of the instrument were set as follows: injection volume was 20 μL, mobile phase was methanol:water = 40:60 (v/v) at the flow rate of 0.7 mL min-1, column temperature was 30 °C and detection wavelength was 270 nm. RESULTS AND DISCUSSION Production of the Recombinant Fluorescent Peptide Tracers. The amino acid and encoding DNA sequences of the fluorescent peptide tracers are shown in Figure S1. The peptidomimetics of imidaclothiz were tethered to EmGFP through the spacers -GGGSGGGS- (for the peptide at the N-terminus) or -GGGSGG- (for the peptide at the C-terminus), in both constructions a 6×His tag was


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included at C terminus for purification. Figure 3a and b shows the SDS-PAGE and western blot analysis of the purified proteins. Homogeneous preparations were obtained for both recombinant tracers after this single step of purification. The C2-15-EmGFP protein migrated with an apparent size of about 30 kDa, which is in agreement with the molecular mass calculated from its amino acid sequence (29685 Da), but the apparent molecular mass of the EmGFP-C2-15 protein was about 2-3 kDa bigger than expected (29800 Da). Although there is no clear explanation for this anomalous migration, a poor processing of the signal peptide (PelB ~2.2 kDa) could account for it. Actually, this could also be one of the reasons why, despite the fact that both recombinant proteins were present in soluble form in the periplasm, the yield of EmGFP-C2-15 (0.5 mg per liter) was much lower than that of C2-15-EmGFP (6.2 mg per liter), but this was not further investigated. Gel filtration experiments showed a single peak for both proteins, discarding the formation of multimers (not shown). The spectra of the fluorescent peptide tracers showed the maximum excitation/emission wavelengths at 485/510 nm (Figure 3c), which, as expected, agrees with the corresponding spectrum of EmGFP. Characterization of the Tracer Reactivity. The reactivity of the recombinant proteins with the mAb 1E7 and its inhibition by imidaclothiz was assayed on ELISA coated with the tracers in a competitive format. As observed in Figure S2, the peptide moiety of the tracers immobilized on the plate reacted with the antibody regardless of its position at the N- or C-terminus of EmGFP. The concentration of imidaclothiz that caused 50% inhibition was similar for both recombinant proteins, with IC50 values of 29.5 and 24.2 ng mL-1 for C2-15-EmGFP and EmGFP-C2-15, respectively. The binding of the fluorescent peptide tracers and mAb 1E7 was further studied by Surface Plasmon Resonance using a Biacore T200 system with a CM7 chip (Figure 4). The KD (KD = koff / kon) of the reaction between



mAb 1E7 and the fluorescent peptide tracers were in the micromolar range, KD = 2.33×10-7 M for C2-15-EmGFP and 0.80×10-7 M for EmGFP-C2-15. In spite of its slightly higher KD, C2-15-EmGFP was chosen to develop the fluorescence immunoassay because it can be produced with higher yields. Development of the MSFIA. In the initial experiments, we noticed that when C2-15-EmGFP was bound to the antibody immobilized on the MNPs, its fluorescence was quenched. This is because the absorption spectrum of Fe3O4 MNPs covers the whole range of wavelengths of visible light, which certainly overlaps the emission wavelength of the fluorescent peptide tracer (Figure 5a). This prevents the measurement of changes in the amount of MNPs-bound tracer due to variations in the concentration of the pesticide. However, once the MNPs are separated from the excess of reagents and incubated with fresh buffer, the fast dissociation of the peptide-mAb 1E7 interaction allows measuring the amount of tracer that dissociates from the MNPs. Indeed, as shown in Figure 5b and c, the fluorescence signals of MNPs suspension and its supernatant begin increasing after the MNPs start being incubated in PBS, reaching a steady state after 25 min. The time course of the accumulation of the fluorescent tracers in the supernatant was also revealed by western blot analysis of the supernatants (Figure 5d). This strategy was used to develop the MSFIA as described in the Methods section, which is schematized in Figure 2. After optimizing the amount of mAb and tracer by checkerboard titration, the inhibition curve displayed in Figure 6 was obtained. The IC50 value, limit of detection (LOD) and linear range (IC10 to IC90) of the MSFIA were 11.0 ng mL-1, 1.87 ng mL-1 and 1.87 to 66.0 ng mL-1, respectively. The IC50 value of the MSFIA was only slightly higher than that of phage ELISA (4.00 ng mL-1),16 which is striking considering that in the latter there is a huge signal amplification caused by


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the strong activity of the enzymatic tracer accumulated along the large surface of the phage. No matter that, the sensitivity of MSFIA is still better than most imidaclothiz immunoassays that use the chemically synthesized hapten as competitor,18-21 and apt for the detection of imidaclothiz below the maximum residue limits (MRL) permitted in China (GB 2763-2014) for agricultural products, such as tea, orange, brown rice and wheat which are 0.3, 0.2, 0.1 and 0.2 mg kg-1, respectively. Cross-Reactivity of MSFIA. The specificity of the test was assessed by measuring the percentage of cross-reactivity with a panel of imidaclothiz related compounds (Table 1). As expected, imidacloprid, which has the same nitro-dihydroimidazol-amine group, showed a significant cross-reactivity of 91.4%, other analogues show negligible CRs (≤ 2.8%). The cross-reactivity pattern was similar to that obtained with the phage ELISA developed with the phage display peptide C2-15. Recovery of Imidaclothiz from Spiked Samples. Considering that methanol is a common component of the solvent mixture used to extract the pesticide, we first studied its possible interference in the assay. Unfortunately, the assay was highly sensitive to the presence of the solvent and started to be affected when its concentration was higher than 2.5% (Figure S3). For this reason, the sample extracts were diluted to bring the methanol below this limit. Matrix interference was studied by performing standard inhibition curves using different dilutions of the matrix extracts (Figure S4). In all cases, after proper dilution the standard curves in diluted matrix were similar to that obtained with 2.5% methanol-PBS buffer, and thus avoiding all matrix effects. Paddy water could be analyzed directly after mixing with an equal volume of 2× PBS buffer. On the other hand, soil and cucumber were analyzed at a final dilution of 20-fold, and at a 40-fold dilution in the case of brown rice. Using these dilution factors, the average recoveries of



imidaclothiz in spiked samples were in the 75.3%-114% range and the RSDs in the 6.1%-11.8% range for all spiking conditions (Table 2). Correlation of MSFIA with HPLC. The cucumber samples were used to compare the data obtained by both methods. The pretreatment method of these samples for HPLC analysis was optimized to remove matrix interference as shown in Figure S5. The average recoveries of HPLC for samples spiked at the concentrations of 40, 200 and 1000 ng g-1 were in the 84.4% to 93.3% range and the RSDs between 3.6% and 3.9% (Table S1). Once the method was optimized, the concentration of imidaclothiz was detected by HPLC and MSFIA in real samples (Table S2). There was a very good correlation with a P value (P = 0.531) higher than 0.05, and a slop value very close to 1 (Figure 7), showing the analytical robustness of the MSFIA for the detection of imidaclothiz in environmental and agricultural samples. CONCLUSIONS In this paper, we fused a peptidomimetic of imidaclothiz with the EmGFP fluorescent protein to construct a novel fluorescent peptide tracer that could be used in combination with MNPs. The recombinant tracer: a) overcomes the limitations of the phage particles as immunoassay reagents, b) provides an assay component with a defined stoichiometry that eliminates the need of chemical conjugating of haptens, and the use of secondary reagents for detection, c) shortens the assay time, and d) enables the development of immunoassays with good analytical performance and low cost. In spite of the lack of an amplification step, the sensitivity of the imidaclothiz MSFIA was high and the test performed with good recoveries in different matrices and good correlation with HPLC. Considering that the isolation of analyte peptidomimetics is a well-established and


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straightforward procedure, the strategy reported here could be applied to any analyte. Perhaps, the only prerequisite would be that the dissociation rate of the peptide-antibody complex be fast, in order to reduce the incubation time before reading. Fortunately, this would be the case for many of the peptidomimetics isolated from phage libraries,13 and they selection can be forced using passive elution during the panning steps. With the growing demand for the detection of small analytes, such as pesticides, environmental pollutants, drugs, metabolites, and so on, we believe that the recombinant fluorescent peptide tracer and MSFIA will be the useful addition to the analytical tool-box for these compounds. AUTHOR INFORMATION Corresponding Author * Tel.: +86 25 84395479. Fax: +86 25 84395479. E-mail address: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31772194), the Fundamental Research Funds for the Central Universities (KYZ201618), the National Key Research and Development Program of China (2017YFF0210200), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17_0572) and CSIC 149 UdelaR Uruguay. ASSOCIATED CONTENT Supporting Information The detailed data of average recoveries of samples spiked with imidaclothiz using HPLC, and the



imidaclothiz residues determined by MSFIAs and HPLC in cucumber samples; the amino acid and DNA sequences of fluorescent peptide tracers; the effect of methanol on MSFIA; the matrix interference on MSFIA; the chromatograms for recoveries of samples spiked with imidaclothiz using HPLC. REFERENCES (1) Arévalo, F. J.; González-Techera, A.; Zon, M. A.; González-Sapienza, G.; Fernández, H. Biosens. Bioelectron. 2012, 32, 231−237. (2) Kim, H. J.; Gonzalez-Techera, A.; Gonzalez-Sapienza, G. G.; Ahn, K. C.; Gee, S. J.; Hammock, B. D. Environ. Sci. Technol. 2008, 42, 2047−2053. (3) Hua, X. D.; Zhou, L. L.; Feng, L.; Ding, Y.; Shi, H. Y.; Wang, L. M.; Gee, S. J.; Hammock, B. D.; Wang, M. H. Anal. Chim. Acta 2015, 890, 150−156. (4) Wang, J.; Liu, Z. P.; Li, G. Q.; Li, J.; Kim, H. J.; Shelver, W. L.; Li, Q. X.; Xu, T. Anal. Bioanal. Chem. 2013, 405, 9579–9583. (5) Kim, H. J.; McCoy, M.; Gee, S. J.; Gonzalez-Sapienza, G. G.; Hammock, B. D. Anal. Chem. 2011, 83, 246–253. (6) Rossotti, M. A.; Carlomagno, M.; González-Techera, A.; Hammock, B. D.; Last, J.; Gonzalez-Sapienza, G. Anal. Chem. 2010, 82, 8838–8843. (7) González-Techera, A.; Vanrell, L.; Last, J. A.; Hammock, B. D.; González-Sapienza, G. Anal. Chem. 2007, 79, 7799–7806. (8) Cardozo, S.; González-Techera, A.; Last, J. A.; Hammock, B. D.; Kramer, K.; González-Sapienza, G. G. Environ. Sci. Technol. 2005, 39, 4234−4241. (9) Hwang, H. J.; Ryu, M. Y.; Park, C. Y.; Ahn, J.; Park, H. G.; Choi, C.; Ha, S. D.; Park, T. J.; Park, J. P. Biosens. Bioelectron. 2017, 87, 164−170. (10) Vanrell, L.; Gonzalez-Techera, A.; Hammock, B. D.; Gonzalez-Sapienza, G. Anal. Chem. 2013, 85, 1177−1182. (11) Yeh, C. Y.; Hsiao, J. K.; Wang, Y. P.; Lan, C. H.; Wu, H. C. Biomaterials 2016, 99, 1−15.


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Table 1. IC50 Values and Cross-Reactivity of a Set of Analogues Structurally Related to Imidaclothiz by MSFIA IC50 (ng mL-1)

CR (%)

Imidaclothiz

11.00

100

Imidacloprid

12.04

91.4

Thiacloprid

387.12

2.8

Clothianidin

2493.50

0.4

Acetamiprid

1763.81

0.6

Thiamethoxam

9605.64

0.1

Nitenpyram

>100000

100000

A Recombinant Fluorescent Peptidomimetic Tracer for

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