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1.
Biosensors (Basel) ; 12(11)2022 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-36421176

RESUMO

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.


Assuntos
Coloide de Ouro , Piretrinas , Animais , Coloide de Ouro/química , Imunoensaio/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Agricultura , Mamíferos
2.
Anal Bioanal Chem ; 414(24): 7143-7151, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36006431

RESUMO

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.


Assuntos
Biotina , Peptidomiméticos , Acrilatos , Benzotiazóis , Fluorimunoensaio/métodos , Ligantes , Peptídeos/química , Sensibilidade e Especificidade , Estreptavidina
3.
Anal Chem ; 94(20): 7358-7367, 2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35536756

RESUMO

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.


Assuntos
Colorimetria , Peptídeos , Cromatografia de Afinidade , Imunoensaio/métodos , Limite de Detecção , Peptídeos/química , Smartphone
4.
Sci Total Environ ; 830: 154690, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35318054

RESUMO

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.


Assuntos
Anticorpos Monoclonais , Espectrometria de Massas em Tandem , Compostos de Anilina , Animais , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática/métodos , Haptenos , Imunoensaio , Camundongos
5.
Biosens Bioelectron ; 201: 113968, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35007993

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Peptidomiméticos , Imunoensaio , Luciferases , Sistemas Automatizados de Assistência Junto ao Leito
6.
J Hazard Mater ; 425: 128011, 2022 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-34896720

RESUMO

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.


Assuntos
Bacteriófagos , Biblioteca de Peptídeos , Bacteriófagos/genética , Ensaio de Imunoadsorção Enzimática , Guanidinas , Imunoensaio , Neonicotinoides , Peptídeos , Sensibilidade e Especificidade , Tiazóis
7.
J Immunol Methods ; 500: 113184, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34808129

RESUMO

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.


Assuntos
Anticorpos Monoclonais/metabolismo , Fusão Celular/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Organotiofosfatos/imunologia , Triazóis/imunologia , Animais , Anticorpos Monoclonais/genética , Ligação Competitiva , Linhagem Celular , Reações Cruzadas , Eletricidade , Ensaios Enzimáticos , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
8.
J Agric Food Chem ; 69(43): 12654-12660, 2021 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-34695356

RESUMO

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.


Assuntos
Microssomos Hepáticos , Espectrometria de Massas em Tandem , Animais , Simulação de Acoplamento Molecular , Compostos Organofosforados , Ratos , Estereoisomerismo , Tiazolidinas
9.
Anal Chem ; 93(34): 11800-11808, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34415158

RESUMO

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.


Assuntos
Bacteriófagos , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Fragmentos de Imunoglobulinas , Biblioteca de Peptídeos
10.
Food Chem ; 360: 130020, 2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34000636

RESUMO

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.


Assuntos
Acrilatos/análise , Benzotiazóis/análise , Dendrímeros/química , Fluoresceína-5-Isotiocianato/química , Imunoensaio de Fluorescência por Polarização/métodos , Peptídeos/química
11.
Sci Total Environ ; 753: 141950, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-32906044

RESUMO

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.


Assuntos
Herbicidas , Água , Ácido 2,4-Diclorofenoxiacético , Animais , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Herbicidas/análise , Coelhos , Espectrometria de Massas em Tandem
12.
Mikrochim Acta ; 188(10): 356, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34585287

RESUMO

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.


Assuntos
Ouro
13.
Front Chem ; 8: 615594, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33344425

RESUMO

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.

14.
Sci Total Environ ; 723: 137909, 2020 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-32222498

RESUMO

Imidacloprid is the most widely used neonicotinoid insecticide and has been reported to pose a threat to ecological security and human health. Therefore, simple-to-operate and highly sensitive methods for the detection of trace levels of imidacloprid are necessary. Here, we isolated two phage-borne peptides that compete with imidacloprid to bind the monoclonal antibody (mAb) 3D11 from phage display peptide libraries. A phage-enzyme-linked immunosorbent assay (P-ELISA) and two phage time-resolved fluoroimmunoassays (P-TRFIAs) for the detection of imidacloprid were developed using the phage-borne peptides as substitutes for chemically synthesized antigens. After systematic optimization, the half-maximum inhibition concentrations (IC50) of the P-ELISA, P-TRFIA-1, and P-TRFIA-2 were 0.067 ng mL-1, 0.085 ng mL-1, and 0.056 ng mL-1, respectively. Based on their IC50 values, the sensitivities of the P-ELISA and P-TRFIAs were more than four times greater than those of previous immunoassays. Additionally, the immunoassays showed satisfactory recovery in the detection of spiked samples and good correlation with high performance liquid chromatography (HPLC) for the detection of samples containing incurred residues.


Assuntos
Bacteriófagos , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoensaio , Neonicotinoides , Nitrocompostos , Peptídeos
15.
Eur J Med Chem ; 193: 112206, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32203787

RESUMO

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.


Assuntos
Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Microssomos Hepáticos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Epóxido Hidrolases/metabolismo , Humanos , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
16.
Sci Total Environ ; 708: 134614, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31806319

RESUMO

In this work, a fluorescent nanoparticles labeling-free fluorescence enzyme-linked immunoassay (FELISA) has been established for the ultrasensitive detection of microcystin-LR (MC-LR) in water and fish samples. Polyclonal antibody against MC-LR was labeled with horseradish peroxidase (HRP) and used as signal probe for binding with analyte in sample or for coating antigen. After washing of the unbound antibody, the substrate system (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS)/H2O2) was added. The oxidation product of ABTS (ox-ABTS) catalyzed by HRP effectively caused the fluorescence quenching of subsequently added silane-doped carbon dots (Si-CDs), and the change in fluorescence intensity of Si-CDs was used to realize the quantitative detection of MC-LR. Under the optimum conditions, the Si-CDs based FELISA method showed a good linear relationship from 0.001 to 3.20 µg L-1 (R2 = 0.994) and provided a low detection limit of 0.6 ng L-1, which was approximately 30-fold lower than that of traditional indirect competitive ELISA. Average recovery values from 79.9% to 109.2% was obtained from spiked water and crucian samples, suggesting its potential application on the monitoring of MR-LR at a trace level.


Assuntos
Água , Animais , Carbono , Carpas , Ensaio de Imunoadsorção Enzimática , Fluorescência , Peróxido de Hidrogênio , Toxinas Marinhas , Microcistinas , Silanos
17.
RSC Adv ; 11(1): 517-524, 2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-35423028

RESUMO

The contents of both pesticide residues and dextran are important parameters for evaluating the quality of sugarcane. In this study, a multicolor upconversion fluorescence immunoassay for the simultaneous detection of thiamethoxam and dextran was established on the basis of magnetic separation. Antigens of thiamethoxam and dextran were coupled to magnetic nanoparticles as the separation elements. Monoclonal antibodies of thiamethoxam (6C7D12) and dextran (3C6F7) were conjugated with the upconversion nanoparticles of NaYF4:Yb,Er with an emission wavelength at 544 nm and NaYF4:Yb,Tm with an emission wavelength at 477 nm to prepare the signaling elements, respectively. Due to the difference in the emission wavelength, the signaling elements bound on the separation elements could be detected simultaneously after separation by an external magnetic field. After optimization, the half-maximal inhibitory concentration (IC50) values of the immunoassay for thiamethoxam and dextran were 0.46 and 49.33 ng mL-1, respectively. The assay showed no cross-reactivity with the analogs of thiamethoxam and dextran except for clothianidin (8.7%). The average recoveries of thiamethoxam and dextran in sugarcane juice were 82.9-93.3% and 87.5-97.2%, respectively. The results indicated that the immunoassay could meet the requirements for the simultaneous quantitative detection of thiamethoxam and dextran.

18.
ACS Appl Mater Interfaces ; 11(36): 33380-33389, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31433617

RESUMO

Peptides isolated from phage display libraries are powerful reagents for small-molecule immunoassay; however, their application as phage-borne peptides is significantly limited by the biological nature of the phage. Here, we present the use of lysine scaffold to prepare a series of different valence peptides to serve as replacements for phage-borne peptides. Benzothiostrobin was selected as a model analyte, the cyclic benzothiostrobin-peptidomimetic in the form of monomer, dendrimer-like dimer, and tetramer were designed and synthesized. Compared with the monomer, the affinity of dendrimer-like dimer and tetramer increased 1.87 and 13.6 times, respectively, as determined by isothermal titration calorimetry (ITC). A novel inner filter effect immunoassay (IFE-IA) with positive readout was developed for benzothiostrobin detection utilizing the peptidomimetics attached to upconversion nanoparticles (UCNPs) as energy donor and monoclonal antibody (mAb)-labeled urchin-like gold nanoflowers (AuNFs) as energy absorber, respectively. The sensitivity of the assay based on dendrimer-like tetramer was approximately 6 and 3 times higher than monomer and dendrimer-like dimer, respectively. After optimization, 50% saturation of the signal (SC50) and detection range (SC10 to SC90) of the IFE-IA based on dendrimer-like tetramer were 11.81 ng mL-1 and 2.04-106.17 ng mL-1, respectively. The IFE-IA also shows good accuracy for the detection of benzothiostrobin in authentic samples.


Assuntos
Dendrímeros/química , Nanopartículas Metálicas/química , Peptídeos/química , Acrilatos/química , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Benzotiazóis/química , Ouro/química , Imunoensaio , Nanopartículas Metálicas/ultraestrutura , Peptidomiméticos , Reprodutibilidade dos Testes , Espectrometria de Fluorescência
19.
Environ Sci Pollut Res Int ; 26(23): 23471-23479, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31197672

RESUMO

A rapid and sensitive immunoassay for the simultaneous detection of imidacloprid and thiacloprid was developed by using magnetic nanoparticles (MNPs) and upconversion nanoparticles (UCNPs). The UCNPs of NaYF4:Yb, Er and NaYF4:Yb, Tm were synthesized and conjugated with anti-imidacloprid monoclonal antibody (mAb) and anti-thiacloprid mAb as signal labels, while the MNPs were conjugated with antigens of thiacloprid and imidacloprid as separation elements. The fluorescence intensities of Yb/Er- and Yb/Tm-doped UCNPs were detected simultaneously in 544 nm and 477 nm under the excitation of NIR light (980 nm). The amounts of mAb-conjugated UCNPs that were separated by antigen-conjugated MNPs were determined based on competitive immunoassays. Under the optimal conditions, the 50% inhibiting concentration (IC50) and limit of detection (LOD, IC10) were 5.80 and 0.32 ng/mL for imidacloprid and 6.45 and 0.61 ng/mL for thiacloprid, respectively. The immunoassay exhibited negligible cross-reactivity with analogs of imidacloprid and thiacloprid except imidaclothiz (86.2%). The average recoveries of imidacloprid and thiacloprid in environmental and agricultural samples, including paddy water, soil, pears, oranges, cucumbers, and wheat, ranged from 78.4 to 105.9% with relative standard deviations (RSDs) of 2.1-11.9% for imidacloprid and ranged from 82.5 to 102.3% with RSDs of 1.0-16.5% for thiacloprid. In addition, the results of the immunoassay correlated well with high-performance liquid chromatography for the detection of the authentic samples.


Assuntos
Imunoensaio/métodos , Nanopartículas de Magnetita/química , Neonicotinoides/análise , Nitrocompostos/análise , Tiazinas/análise , Fluorescência , Magnetismo , Nanopartículas/química , Tiazóis
20.
Environ Int ; 127: 694-703, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30991225

RESUMO

Isofenphos-methyl (IFP) is a very active and persistent chiral insecticide. However, IFP has lower activity against acetylcholinesterases (AChEs). Previously, it was confirmed that phosphorothioate organophosphorus pesticides with N-alkyl (POPN) require activation by oxidative desulfuration and N-dealkylation. In this work, we demonstrated that IFP could be metabolized in human liver microsomes to isofenphos-methyl oxon (IFPO, 52.7%), isocarbophos (ICP, 14.2%) and isocarbophos oxon (ICPO, 11.2%). It was found that (R)-IFP was preferentially degraded compared to the (S)-enantiomer, and the enantiomeric fraction (EF) value reached 0.61 at 60 min. However, (S)-enantiomers of the three metabolites, were degraded preferentially, and the EF values ranged from 0.34 to 0.45. Cytochrome P450 (CYP) isoforms CYP3A4, CYP2E1, and CYP1A2 and carboxylesterase enzyme have an essential role in the enantioselective metabolism of IFP; but, the enzymes that participate in the degradation of IFP metabolites are different. The AChE inhibition bioassay indicated that ICPO is the only effective inhibitor of AChE. The covalent molecular docking has proposed that the metabolites of IFP and its analogs after N-dealkylation and oxidative desulfuration will possess the highest inhibitory activity against AChE. This study is the first to demonstrate that ICPO can be regarded as a potential biomarker for the biomonitoring of IFP and ICP exposure in humans.


Assuntos
Compostos Organotiofosforados/metabolismo , Biomarcadores/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Humanos , Malation/análogos & derivados , Microssomos Hepáticos , Simulação de Acoplamento Molecular , Compostos Organotiofosforados/química , Estereoisomerismo
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