RESUMEN

Organophosphorus pesticides (OPs) are widely used in agriculture and the monitoring of their residues is very important to protect human health. Immunoassays are important tools for the analysis of small molecules. Generally, noncompetitive mode of immunoassay is considered to be more sensitive than competitive mode. In this study, peptides that can identify immunocomplex of OPs were screened from a phage display library. Subsequently, a second-generation peptide library was constructed and peptides with better performance were isolated. Then, a rapid and sensitive noncompetitive magnetic-phage anti-immunocomplex assay (MPHAIA) for OPs was developed based on the best phage-peptide and single chain antibody immunomagnetic beads. The MPHAIA showed broad specificity for OPs with a thiophosphate group. The half-saturated concentration (SC50) values and limits of detection (LODs) of MPHAIA to 12 OPs were ranged from 15.04 to 105.48 ng/mL and 4.07-14.19 ng/mL, respectively. The accuracy and reliability of MPHAIA were verified by gas chromatography-tandem mass spectrometry (GC-MS/MS) parallel analysis of six kinds of OPs in spiked cucumber samples. The recovery rates were in range of 81.2-116.3% with coefficient of variation from 4.1% to 14.1%, which were consistent with the results of GC-MS/MS.

Asunto(s)

RESUMEN

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.

RESUMEN

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.

Asunto(s)

RESUMEN

The development of immunosensors for the detection of small molecules is of great interest because of their simplicity, high sensitivity and extended analytical range. Due to their size, small compounds cannot be simultaneously recognized by two antibodies impeding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. In this work, we combine the advantages of magneto-electrochemical immunosensors with the improved sensitivity and direct proportional signal of noncompetitive immunoassays to develop a new Phage Anti-Immunocomplex Electrochemical Immunosensor (PhAIEI) for the detection of the herbicide atrazine. The noncompetitive assay is based on the use of recombinant M13 phage particles bearing a peptide that specifically recognizes the immunocomplex of atrazine with an anti-atrazine monoclonal antibody. The PhAIEI performed with a limit of detection (LOD) of 0.2 pg mL(-1), which is 200-fold better than the LOD obtained using the same antibody in an optimized conventional competitive ELISA, with a large increase in working range. The developed PhAIEI was successfully used to assay undiluted river water samples with no pretreatment and excellent recoveries. Apart from the first demonstration of the benefits of integrating phage anti-immunocomplex particles into electrochemical immunosensors, the extremely low and environmentally relevant detection limits of atrazine attained with the PhAIEIS may have direct applicability to fast and sensitive detection of this herbicide in the environment.

Asunto(s)