Active Ester Method Research Articles

Tailored design of NHS–SS–NHS cross-linked chitosan nano-hydrogels for enhanced anti-tumor efficacy by GSH-responsive drug release

The traditional chemotherapeutic agents’ disadvantages such as high toxicity, untargeting and poor water solubility lead to disappointing chemotherapy effects, which restricts its clinical application. In this work, novel size-appropriate and glutathione (GSH)-responsive nano-hydrogels were successfully prepared via the active ester method between chitosan (containing –NH2) and cross-linker (containing NHS). Especially, the cross-linker was elaborately designed to possess a disulfide linkage (SS) as well as two terminal NHS groups, namely NHS–SS–NHS. These functionalities endowed chitosan-based cross-linked scaffolds with capabilities for drug loading and delivery, as well as a GSH-responsive mechanism for drug release. The prepared nano-hydrogels demonstrated excellent performance applicable morphology, excellent drug loading efficiency (∼22.5%), suitable size (∼100 nm) and long-term stability. The prepared nano-hydrogels released over 80% doxorubicin (DOX) after incubation in 10 mM GSH while a minimal DOX release less than 25% was tested in normal physiological buffer (pH = 7.4). The unloaded nano-hydrogels did not show any apparent cytotoxicity to A 549 cells. In contrast, DOX-loaded nano-hydrogels exhibited marked anti-tumor activity against A 549 cells, especially in high GSH environment. Finally, through fluorescent imaging and flow cytometry analysis, fluorescein isothiocyanate-labeled nano-hydrogels show obvious specific binding to the GSH high-expressing A549 cells and nonspecific binding to the GSH low-expressing A549 cells. Therefore, with this cross-linking approach, our present finding suggests that cross-linked chitosan nano-hydrogel drug carrier improves the anti-tumor effect of the A 549 cells and may serve as a potential injectable delivery carrier.

Active ester-based peptide bond formation and its application in peptide synthesis

Active ester method is an efficient strategy to address the notorious racemization/epimerization issue of peptide bond formation. Herein, the pros and cons of using active esters for peptide synthesis were systematically summarized and analyzed.

Preparation of Monoclonal Antibodies Specifically Reacting with the Trichothecene Mycotoxins Nivalenol and 15-Acetylnivalenol via the Introduction of a Linker Molecule into Its C-15 Position.

Nivalenol (NIV) is a trichothecene mycotoxin that is more toxic than deoxynivalenol. It accumulates in grains due to infection with Fusarium species, which are the causative agents of scab or Fusarium head blight. An immunoassay, which is a rapid and easy analytical method, is necessary for monitoring NIV in grains. However, a specific antibody against NIV has not been prepared previously. To establish an immunoassay, we prepared NIV, introduced a linker, and generated antibodies against it. NIV was prepared from a culture of Fusarium kyushuense obtained from pressed barley through chromatographic procedures with synthetic adsorbents and silica gel. NIV was reacted with glutaric anhydride, and the reaction was stopped before mono-hemiglutaryl-NIV was changed to di-hemiglutaryl-NIV. 15-O-Hemiglutaryl-NIV was isolated via preparative HPLC and bound to keyhole limpet hemocyanin (KLH) using the active ester method. Two different monoclonal antibodies were prepared by immunizing mice with the NIV-KLH conjugate. The 50% inhibitory concentration values were 36 and 37 ng/mL. These antibodies also showed high reactivity in a direct competitive enzyme-linked immunosorbent assay and specifically reacted with NIV and 15-acetyl-NIV but not with deoxynivalenol and 4-acetyl-NIV.

Rapid determination of 2,4-diaminopyrimidine residues through sample pretreatment using immunomagnetic bead purification along with HPLC–UV

To reduce matrix interference and realize simultaneous detection of multiple homologous compounds (trimethoprim (TMP), diaveridine (DVD), ormetoprim (OMP), baquiloprim (BQP), and aditoprim (ADP) in pig, cattle, chicken, and fish muscles), an immunomagnetic bead (IMB)-based sample purification pretreatment with HPLC–UV was developed. A broad-spectrum monoclonal antibody (mAb, named 14C6) was prepared and conjugated with carboxylic-acid-functionalized magnetic nanoparticles using the active ester method to obtain IMBs for sample purification. The extraction solvent was optimized based on the extraction efficiency. Good linearity was observed for all the five analytes (10–200 μg/kg) with the LOD and LOQ of 5 and 10 μg/kg, respectively. The mean recoveries ranged from 62.5% to 76.9%, while the coefficient of variation was <12.2%. The IMB method afforded greater sample purification and enrichment than those achieved with the SPE column-based conventional method. Hence, the IMB-based sample purification is a useful tool to determine 2,4-diaminopyrimidine residues in edible animal tissues.

A high-performance fluorescence immunoassay based on pyrophosphate-induced MOFs NH2-MIL-88B(Fe) hydrolysis for chloramphenicol detection

In this work, we firstly found and verified that pyrophosphate (PPi) can form a competitive coordination effect with the metal-organic frameworks (MOFs) NH2-MIL-88B(Fe), causing the MOFs structure to collapse and release diaminoterephthalic acid (NH2-BDC), and then weakening the ligand-metal charge transfer (LMCT) of the MOFs network structure, allowing the original fluorescence of NH2-BDC be recovered. Based on this new principle, synthesized high-stability MOFs NH2-MIL-88B(Fe) were employed as fluorescent signal sources to trace target recognition, and MOFs fluorescent immunoprobes were prepared by chemically coupling anti-chloramphenicol (CAP) monoclonal antibodies via the active ester method (EDC/Sulfo-NHS). A direct competitive fluorescence immunoassay was established for the detection of CAP residues in milk samples, in which the limit of detection (LOD) was 0.028 μg L−1, the linear range was 0.05–0.75 μg L−1, and the recovery rate was 91.8%–112.3%. This study employed a simple and time-saving one-step method for releasing fluorescent signals, which was also useful for the rapid and accurate detection of CAP residues.

Development of a New Monoclonal Antibody against Brevetoxins in Oyster Samples Based on the Indirect Competitive Enzyme-Linked Immunosorbent Assay.

The consumption of shellfish contaminated with brevetoxins, a family of ladder-frame polyether toxins formed during blooms of the marine dinoflagellate Karenia brevis, can cause neurotoxic poisoning, leading to gastroenteritis and neurotoxic effects. To rapidly monitor brevetoxin levels in oysters, we generated a broad-spectrum antibody against brevetoxin 2 (PbTx-2), 1 (PbTx-1), and 3 (PbTx-3) and developed a rapid indirect competitive enzyme-linked immunosorbent assay (icELISA). PbTx-2 was reacted with carboxymethoxylamine hemihydrochloride (CMO) to generate a PbTx-2-CMO hapten and reacted with succinic anhydride (HS) to generate the PbTx-2-HS hapten. These haptens were conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to prepare immunogen and coating antigen reagents, respectively, using the active ester method. After immunization and cell fusion, a broad-spectrum monoclonal antibody (mAb) termed mAb 1D3 was prepared. The 50% inhibitory concentration (IC50) values of the icELISA for PbTx-2, PbTx-1, and PbTx-3 were 60.71, 52.61, and 51.83 μg/kg, respectively. Based on the broad-spectrum mAb 1D3, an icELISA was developed to determine brevetoxin levels. Using this approach, the limit of detection (LOD) for brevetoxin was 124.22 μg/kg and recoveries ranged between 89.08% and 115.00%, with a coefficient of variation below 4.25% in oyster samples. These results suggest that our icELISA is a useful tool for the rapid monitoring of brevetoxins in oyster samples.

Quantum dots‐based fluorescence immunoassay for detection of tiamulin in pork

AbstractTiamulin (TML) is a kind of diterpenoid veterinary antibiotic. If abused, it will cause residues in edible tissues of animals and endanger human health. Thus, a facile, sensitive, and accurate method is essential for TML detection. In this study, TML complete antigen was synthesized by oximation and active ester method, and monoclonal antibodies with high specificity and sensitivity were prepared. ZnCdSe/ZnS quantum dots (QDs) coupled with goat anti‐mouse IgG were used as a fluorescence detection probe to establish indirect competitive fluorescent immunosorbent assay (icFLISA) for the determination of TML in pork. The 50% inhibitory concentration (IC50) of the icFLISA was 0.069 ng/ml, and the limits of detection (LOD) were 0.309 pg/ml. The recovery of the spiked pork samples for the icFLISA ranged from 85.6 to 96.8%, and the coefficient of variation (CV) was less than 9.4%. The results showed that the icFLISA based on ZnCdSe/ZnS QDs was more sensitive than the traditional indirect competitive enzyme‐linked immunosorbent assay for the detection of TML in edible animal tissues.

Immunochromatographic assay based on high-affine monoclonal antibody for the detection of imidocarb in milk.

Imidocarb (IM) is an antiprotozoal agent, which is mainly used to treat babesiosis and piroplasmosis for animals. However, overdose or improbable utilization cause IM residues in animal origin products, which would be harmful to human health. Here, a monoclonal antibody (mAb) against IM with extremely sensitive and specific features has been successfully prepared from a novel immunogen synthesized by virtue of the active ester method. The concentration of half-maximal inhibition (IC50 ) of the mAb was 0.074ng/ml and the affinity constant was 4.58 × 1010 L/mol. On the basis of this condition, an immunochromatographic strip (ICS) is proposed that could be applied in milk samples to test IM rapidly. For the ICS, the visual detection limit (cut-off value) was 5ng/ml, IC50 was 0.4ng/ml, the limit of detection (LOD) was 0.078ng/ml, the linear detection scope was 0.117 to 1.37ng/ml. The recovery rates ranged from 88.83% to 91.47% and coefficients of variation (CV) were in the spectrum of 7.31% to 9.43%. In general, the recommended test strip provided an exceedingly simple and reliable detection method for quickly testing the IM. PRACTICAL APPLICATION: In our joint efforts, an extremely sensitive monoclonal antibody against imidocarb was obtained and a test strip for the rapid detection of imidocarb in milk was developed. The developed method could be applied to the field and provided great potential for analytical of imidocarb in other matrixes.

A highly sensitive and dual-readout immunoassay for norfloxacin in milk based on QDs-FM@ALP-SA and click chemistry

A dual-readout immunoassay based on QDs-FM@ALP-SA and click chemistry was developed for quick and sensitive detection of norfloxacin (NOR), which is an important fluoroquinolone antibiotic. In the system, the NOR-biotin conjugate (NOR-Biotin) was synthesized by click chemistry for signal transformation, and alkaline phosphatase–labeled streptavidin (ALP-SA) was attached to quantum dot fluorescence microspheres (QDs-FM) by an activated ester method to form QDs-FM@ALP-SA for signal amplification. Here, QDs-FM was a dual-functional carrier: it was used not only as a chemiluminescence signal amplification carrier but also as a fluorescent signal due to its fluorescence character. The NOR antibody was coated on a 96-well chemiluminescence microtiter plate, and NOR-Biotin was bound to the antibody specifically. Then, QDs-FM@ALP-SA was combined with NOR-Biotin to develop a direct competition chemiluminescence/fluorescence immunoassay (dc-CLIA/FIA). The IC50 values were 0.345 and 1.206 ng/mL for dc-CLIA/FIA, respectively. The linear range was 0.013–12.48 ng/mL and 0.042–39.86 ng/mL, respectively. The recovery from the standard fortified blank milk samples was in the range of 86.44%–101.3%. Therefore, this method could be a useful tool for routine screening of NOR residues in milk.

Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites.

This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 102 in supernatants and (1.28 to 5.12) × 105 in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 μg/L in the supernatants and 18.12 to 31.46 μg/L in the ascites. The affinity constant (Ka) of all of the mAbs was between 4.15 × 109 and 6.54 × 109 L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 μg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 μg/L, and its linear working range was between 1.03 and 288.55 μg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed.

Magnetic particles encoding a suspension probe for ultra-sensitive and quantitative determination of atrazine

As a highly toxic and widely used herbicide, atrazine poses a serious threat to food safety as well as overall environmental and human health. Due to complex matrix interference and the difficulty of signal enrichment, there is an urgent need for a convenient, fast, and ultrasensitive method that detects trace atrazine without concern for matrix effects. Here, we provide the first account of a sensitive and rapid suspension probe based on magnetic microspheres used to detect atrazine in herbs. The self-made magnetic beads featured -COOH groups and were used as the carrier to construct immunofluorescent probes. These probes then conjugated with the atrazine antigen through an activated ester method, ultimately binding to the antibody. Homogeneous detection was ensured using flow cytometry and the microflow optical channel along with allophycocyanin-conjugated goat-anti-mouse secondary antibody (APC-IgG-SecAb) as the fluorescent signal. The magnetic suspension probe allowed for high target enrichment and the inherent two-dimensional selective detection of flow cytometry effectively avoided any matrix interference. This method had good linearity across 1.69−23.19 ng mL−1. The IC50 and LOD values were 4.81 ng mL−1 and 0.95 ng mL−1, respectively; the sensitivity was increased three-fold relative to ELISA. After complete optimization, 2-N-morpholinoeth-anesulfonic acid was used as the coupling solution and maintained good mono-dispersity, stability, and reactivity for the labelled microspheres during the process. The entire experiment was simple, and effectively used reagents; moreover, both the labor required and detection time were greatly reduced. Critically, the strategy presented here greatly reduced interference from complex matrices, and saved preparation for matrix-matched solutions when different herbs were screened. Overall, this strategy was sensitive, rapid, eco-friendly, and labor-saving; collectively, these attributes make it well-suited for on-site screening of atrazine contamination and will allow for increased food safety.

Amino-Acid Derivatives of Pyranocoumarins

A methodology for synthesizing amino-acid derivatives of pyranocoumarins in three steps using pyranocoumarin oximes as starting compounds was developed. A series of pyranocoumarins containing the amino acids glycine, alanine, phenylalanine, methionine, leucine, and β-alanine in their structures could be prepared using the activated ester method.

Colloidal gold-based immunochromatographic strip assay for the rapid detection of diminazene in milk

ABSTRACT Diminazene (DIM) is widely used to treat trypanosomiasis in livestock, but its residues in animal products can cause food safety problems. Thus, a rapid colloidal gold-based immunochromatographic strip assay was established to analyse DIM residue in milk samples. A highly sensitive and specific monoclonal antibody (mAb) against DIM was produced with a new synthetic immunogen by the active ester method. The titre of the prepared antibody was up to 1:1.0 × 106, the affinity constant was 2.2 × 108 L mol−1 and the half-maximal inhibitory concentration (IC50) was 0.4 ng mL−1 based on enzyme-linked immunosorbent assay (ELISA). Then, the colloidal gold-labelled mAb probe was successfully prepared and used for preparing the immunochromatographic strip. The strip showed high sensitivity and specificity, the IC50 for DIM was 5.2 ng mL−1, the limit of detection was 1.2 ng mL−1 and the linear range of detection was 1.8–15.4 ng mL−1. The average recoveries ranged from 89.5% to 91.7%, and coefficients of variation were 6.6–7.2%. The immunochromatographic strip assay can be employed in the rapid detection of DIM residue in milk samples.

Synthesis of N-Acetylmuramyl-L-Alanyld- Isoglutamine α-Phenylglycoside

N-Acetylmuramyl-L-alanyl-D-isoglutamine α-phenylglycoside was synthesized from α-phenyl-D-glucosaminide peracetate, which was obtained by fusing β-D-glucosamine pentaacetate with phenol and zinc chloride followed by deacetylation. α-Phenyl-N-acetylglucosaminide was converted in two steps into 4,6-O-isopropylidene-N-acetyl-D-muramic acid α-phenylglycoside, which was condensed with L-Ala-D-iGln benzyl ether using the activated ester method. Sequential removal of glycopeptide protecting groups via acid hydrolysis and catalytic hydrogenolysis gave the target product.

Synthesis, characterization and bio-functionalization of magnetic nanoparticles to improve the diagnosis of tuberculosis

Mycobacterium tuberculosis is the cause of one of the diseases with the highest mortality and morbidity rate in the Americas and in the world. In developing countries, the diagnosis of tuberculosis (TB) is based on baciloscopy and bacteriological cultures. The first method has a low sensitivity, and the second can take several weeks to reach a confirmatory diagnosis. The lack of a rapid diagnosis compromises the efforts to control this disease and favors the transmission of tuberculosis to the susceptible population. In this work, we present the synthesis, amine-silanization, characterization and bio-functionalization of magnetic nanoparticles (MNPs) to develop a sandwich ELISA to detect and concentrate antigens from M. tuberculosis. For this purpose, a recombinant mycobacterial heat shock protein Hsp16.3, which contributes to the persistence of TB, was cloned and expressed in the E. coli system. Polyclonal antibodies anti-Hsp16.3 were produced in a rabbit and in mice. Magnetic nanoparticles were synthesized by co-precipitation, amine-functionalized and characterized by several physical-chemical methods. The XRD, Mossbauer spectroscopy, zeta potential, TEM, and FTIR all proved the successful preparation of the MNPs showing a diffraction crystal diameter of 10.48 ± 2.56 nm, superficial net charge of : +23.57 ± 2.87 mV, characteristic patterns of magnetite and a structure similar to a sphere. Additionally, it showed a magnetization saturation of 37.06 emu.g−1. For the functionalization of nanoparticle surfaces with anti-Hsp16.3, the active ester method was used for bond formation, and parameters such as time of incubation, coupling agents ratio (EDC/NHS) and concentration as well as surface saturation level of amine-silanized MNPs (MNP@Si@NH2) were standardized. Finally, bio-functionalized MNPs were used to detect, fix and concentrate the recombinant antigen Hsp16.3 from M. tuberculosis in a sandwich ELISA-MNP assay.

Flow-Based Immunosensing Using the Pore Channel of a Porous Membrane As a Reaction Space.

This Letter describes a new rapid and sensitive immunosensing device using the pore space of a porous membrane as the reaction space. A track-etched membrane with uniform cylindrical pores is used as the base substrate of this device. The capture antibodies are covalently and densely immobilized inside the membrane pores by the uniform introduction of poly(acrylic acid) (PAAc) via the plasma graft polymerization technique, followed by the active ester method. This membrane shows excellent antibody retention by covalent binding. The detection test was carried out via a sandwich-type assay, and all reaction steps from the antigen-antibody reaction to the enzyme reaction were conducted by permeating each solution into the pores. The detection test showed a signal comparable to that of the conventional enzyme-linked immunosorbent assay, although the detection time required in the test was shortened to 35 min. The reason for achieving both high sensitivity and short detection time is that the antibody accumulated pore space with high selectivity and promoted contact between the reactants by solution permeation. This report is expected to aid the design of systems for membrane-based devices, which currently have problems associated with sensitivity, rapidity, selectivity, or amount of sample. We further expect that this system could be applied to various diagnostic areas, including point-of-care testing.

Synthesis of Isoflavone–Amino-Acid Conjugates

Conjugates of natural and synthetic amino acids and 7-hydroxyisoflavone in which the amino-acid residue was bonded to the isoflavone through a hydroxyacetate linker were prepared using the activated ester method.

Artificial Esterase Based on Self-assembly Gel Microspheres Constructed from Chitosan and Amino Acids

A novel artificial esterase based on chitosan and amino acids was synthesized in the present study. The Fmoc-His and Glu were linked to chitosan by active ester method(AEM). The hydroxide radical in chitosan, imidazole group of Fmoc-His and carboxyl from Glu formed a catalytic center of natural esterase. Gel microspheres were coated with a protective layer and a supporting layer by self-assembly construction function in carboxymethylcellulose sodium(CMCS) solution. As for catalytic activity, chitosan-His-Glu was found to be more efficient than chitosan- His and chitosan-Glu in mimicking the core catalytic sites of natural esterase, and the best ratio(mass ratio) of chitosan-His-Glu:CMCS was 1:3. Furthermore, metal ions, such as Ca2+, Mg2+, Fe2+, etc., were able to improve the catalytic efficiency of artificial esterase. And the Lineweaver-Burk plot indicated that the catalytic kinetics of artificial esterase conformed to Michaelis-Menten equation.

Quantum Dot-Labeled Immunochromatographic Test Strip for Rapid Detection of Ochratoxin A in Cereals

Objective:A quantum dot-labeled immunochromatographic test strip for the determination of ochratoxin A(OTA)in cereals was developed based on the competitive inhibition principle. Methods:Quantum dot antibody conjugates(QD-Ab)were prepared by coupling an carboxy-functionalized quantum dot(QD)with an ochratoxin A polyclonal antibody(Antibody,Ab)via the activated ester method;Different amounts of QD-Ab and working fluid were used to optimize the working conditions of the quantum dot-labeled immunochromatographic test strip;The effectiveness of the method was verified by a commercial kit. Results:When the molar ratio of QD to Ab was 1:10,the fluorescence characteristics of QD-Ab were the best;when QD-Ab and working fluid were added at 1,10 μL,respectively,the best results of quantum dot-labeled immunochromatographic test strips were obtained;The detection limit of the quantum dot-labeled immunochromatographic test strip in the assay buffer was 0.5 μg/L,and the detection limit of OTA in the cereal sample was 5 μg/kg,the entire detection process could be completed within 10 min;The quantum dot-labeled immunochromatographic test strip had good specificity and effectiveness. Conclusion:This assay is simple,rapid,and sensitivity. It can meet the requirements of on-site rapid detection of ochratoxin A in cereals.

Method for the Synthesis of γ-PEGylated Folic Acid and Its Fluorescein-Labeled Derivative

This paper reports exclusive γ-conjugation of poly(ethylene glycol) (PEG) to folic acid (FA) and the synthesis of its fluorescein (Fl)-labeled derivative. FA is first reacted with 1 equiv of n-BuLi that forms the metal salt exclusively at the γ-position. Model reactions between γ-lithiated FA and bromo-alcohols were used to demonstrate exclusive γ-conjugation. γ-PEGylated FA was then synthesized by reacting γ-lithiated FA with a bromine-functionalized methoxy-PEG (Br-PEGM) that is prepared by Candida antarctica Lipase B (CALB)-catalyzed transesterification of ethyl 5-bromovalerate (EBV) with methoxy-PEG (HO-PEGM). Fl-labeled γ-PEGylated FA was also synthesized by CALB-catalyzed Michael addition of FA-γ-SH to acrylate-PEG-Fl that was made by a series of CALB-catalyzed transformations. NMR spectroscopy and MALDI-ToF spectrometry verified the structure and purity of the compounds. These lithiation-based methods are superior compared to both the currently used activated ester method that gives a mixture of γ,...