Determination of 19-nortestosterone residues in aquaculture tissues by enzyme-linked immunosorbent assay and comparison with liquid chromatography and tandem mass spectrometry

19-Nortestosterone (17β-NT) was oximated by carboxymethoxylamine and then coupled with bovine serum albumin (BSA) in a mixed-anhydride reaction in order to produce an antibody. The conjugate rate of 17β-NT and BSA was estimated to be 24 by ultraviolet spectrophotometry. Polyclonal antibody of 17β-NT was acquired from the animal immunized with the conjugate. Through an indirect enzyme-linked immunosorbent assay (ELISA), which demonstrated that the synthesis of immunogen was successful, the titre of antiserum was found to be 6.4?×?10⁵. Based on the purified antibody, a competitive indirect ELISA was developed. ELISA revealed that the limit of detection (LOD) was 0.07?ng?g^?1, the recovery (in edible tissues) was 71–89%, and the working range was 0.05–31.25?ng?g^?1. The preliminary evaluation of assay performance through specificity, sensitivity, precision, and accuracy revealed that this ELISA method could be used in the practical detection of 17β-NT in tissue samples. Moreover, this method was compared with high-performance liquid chromatography tandem mass spectrometry, for which the transition for quantification of 17β-NT was 275.4/109.1.     

Cu immobilized on chitosan-modified iron oxide magnetic nanoparticles: Preparation,characterization and investigation of its anti-lung cancer effects

Chitosan is a linear polysaccharide and non-toxic bioactive polymer with a wide variety of applications due to its functional properties such as ease of modification, and biodegradability. In this study, a green protocol for supporting of Cu(II) on chitosan-encapsulated magnetic Fe₃O₄ nanoparticles is described. The morphological and physicochemical features of the material were determined using several advanced techniques like fourier transformed infrared spectroscopy (FT-IR), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), inductively coupled plasma (ICP), vibrating sample magnetometer (VSM) and X-ray photoelectron spectroscopy (XPS). The average diameter of the NPs was approximately 15–25 nm. In addition, the Fe₃O_/CS/Cu(II) nanocomposite was engaged in biological assays like study of anti-oxidant properties by DPPH mediated free radical scavenging test using BHT as a reference molecule. Thereafter, on having a significant IC₅₀ value in radical scavenging assay, we extended the bio-application of the desired nanocomposite in anticancer study of lung well-differentiated bronchogenic adenocarcinoma, lung moderately differentiated adenocarcinoma, and lung poorly differentiated adenocarcinoma of human lung in-vitro conditions. In the cytotoxicity and anti-human lung studies, the nanocomposite was treated to lung cancer lung well-differentiated bronchogenic adenocarcinoma (HLC-1), lung moderately differentiated adenocarcinoma (LC-2/ad), and lung poorly differentiated adenocarcinoma (PC-14) cell line following MTT assay. The cell viability of malignant lung cell line reduced dose-dependently in the presence of Fe₃O_/CS/Cu(II) nanocomposite. The recent results suggest that Fe₃O_/CS/Cu(II) nanocomposite have a suitable anticancer activity against lung cell lines.     

High temporal resolution monitoring of enzyme reaction and inhibition using optically gated vacancy capillary electrophoresis and immobilized enzyme

A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5 s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.     

光子晶体生化传感器的构建及应用

光子晶体生化传感分析是基于光子晶体的结构特性将生化信号放大或转换为光电可读信号,并通过仪器或肉眼进行定量或半定量分析的方法.本文围绕着光子晶体生化传感器的材料选择与构建、传感机理、设计及应用三方面进行综述.在材料选择与构建上,本文从单分散胶体颗粒、电磁复合胶体颗粒、纳米线、金属-有机框架等方面进行综述;在传感机理上,本...     

Indole- and Pyrazole-Glycyrrhetinic Acid Derivatives as PTP1B Inhibitors: Synthesis,In Vitro and In Silico Studies

Regulating insulin and leptin levels using a protein tyrosine phosphatase 1B (PTP1B) inhibitor is an attractive strategy to treat diabetes and obesity. Glycyrrhetinic acid (GA), a triterpenoid, may weakly inhibit this enzyme. Nonetheless, semisynthetic derivatives of GA have not been developed as PTP1B inhibitors to date. Herein we describe the synthesis and evaluation of two series of indole- and N-phenylpyrazole-GA derivatives (4a–f and 5a–f). We measured their inhibitory activity and enzyme kinetics against PTP1B using p-nitrophenylphosphate (pNPP) assay. GA derivatives bearing substituted indoles or N-phenylpyrazoles fused to their A-ring showed a 50% inhibitory concentration for PTP1B in a range from 2.5 to 10.1 µM. The trifluoromethyl derivative of indole-GA (4f) exhibited non-competitive inhibition of PTP1B as well as higher potency (IC₅₀ = 2.5 µM) than that of positive controls ursolic acid (IC₅₀ = 5.6 µM), claramine (IC₅₀ = 13.7 µM) and suramin (IC₅₀ = 4.1 µM). Finally, docking and molecular dynamics simulations provided the theoretical basis for the favorable activity of the designed compounds.     

Pinus wallichiana-synthesized silver nanoparticles as biomedical agents: in-vitro and in-vivo approach

ABSTRACT

The current study aims to assess the aqueous extract of Pinus wallichiana stem for the synthesis of small spherical-shaped (10–30?nm) silver nanoparticles (AgNPs) and their in-vitro and in-vivo biomedical applications. The biosynthesized AgNPs were nonmutagenic and safe at all test doses as per Ames and acute toxicity assay (20, 40, 60, and 80?mg/kg). The percent writhing inhibitory effect generated by AgNPs was 42.51, 50.84, and 59.06 at test doses of 10, 20, and 30?mg/kg, respectively. The percent decreased in gastrointestinal tract motility observed was 41.34%, 32.69%, and 28.48% at 10, 20, and 30?mg/kg, respectively. They also showed a significant antipyretic effect after 1, 2, and 3?h in comparison to normal saline. The AgNPs of P. wallichiana showed good antibacterial activity against Acinetobacter baumannii (60% with MIC₅₀?=?2.36?mg/ml and MBC?=?5.0?mg/ml). These nanoparticles also possessed good antioxidant activity of 61.77?±?0.828% and 70.25?±?0.56% at 400 and 500?µg/ml, respectively and lack phytoagglutinin potential. Because of their high potency as biomedical agents, these nanoparticles can be a good alternative to the currently available drugs and approaches.     

Monoclonal antibody-based enzyme-linked immunosorbent assays for the detection of the organophosphorus insecticide diazinon

Four haptens of the organophosphorus (OP) insecticide diazinon were synthesized to develop enzyme-linked immunosorbent assays (ELISAs) for this pesticide. One of them was conjugated to KLH to be used as the immunogen for production of monoclonal antibodies. By using the antibodies and a coating antigen, an indirect competitive ELISA was developed, which showed an IC₅₀ of 4.0 ng/mL with a detection limit of 0.7 ng/mL. A direct competitive ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 6.0 ng/mL with a detection limit of 0.9 ng/mL. The antibodies in both assays showed negligible cross-reactivity with metabolites of diazinon and other OP pesticides. Recovery of diazinon from fortified lettuce and rice samples was satisfactory except at the fortified concentration of 100 ppb.     

基于内滤效应的信号增强型诺氟沙星荧光分析法

基于茜素红和卟啉之间的荧光内滤效应,成功构建了一种分子识别事件与信号报告空间分离的、高选择性的荧光增强型诺氟沙星分析方法。结果表明,无诺氟沙星时,茜素红在420 nm处有最大吸收,这和卟啉的最大激发波长有较大重叠,茜素红和卟啉之间因发生内滤效应导致卟啉的荧光被有效猝灭;而茜素红与诺氟沙星的配合物在523 nm处有最大吸收,和卟啉的最大激发波长不再重叠,即诺氟沙星与茜素红之间的荷移反应破坏了该内滤效应,导致卟啉荧光恢复,据此,可将茜素红的吸收信号转变为高灵敏的卟啉的荧光信号。在最佳实验条件下,诺氟沙星的质量浓度在10 ~ 450 mg?L-1范围内与体系的相对荧光强度(IF/I0F)呈线性关系(r2=0.987 8),检出限(S/N=3)为5 mg?L-1。方法选择性好,常见金属离子和药物辅料不干扰诺氟沙星的测定。该研究利用内滤效应,将灵敏度较低的诺氟沙星紫外可见分析法转换为灵敏度较高的荧光分析法,且无需将分子识别单元和信号转导单元共价连接,无需复杂的荧光探针合成工艺,为设计该类药物的荧光分析法提供了新思路。     

Tracing Binding Modes in Hit‐to‐Lead Optimization: Chameleon‐Like Poses of Aspartic Protease Inhibitors

Successful lead optimization in structure‐based drug discovery depends on the correct deduction and interpretation of the underlying structure–activity relationships (SAR) to facilitate efficient decision‐making on the next candidates to be synthesized. Consequently, the question arises, how frequently a binding mode (re)‐validation is required, to ensure not to be misled by invalid assumptions on the binding geometry. We present an example in which minor chemical modifications within one inhibitor series lead to surprisingly different binding modes. X‐ray structure determination of eight inhibitors derived from one core scaffold resulted in four different binding modes in the aspartic protease endothiapepsin, a well‐established surrogate for e.g. renin and β‐secretase. In addition, we suggest an empirical metrics that might serve as an indicator during lead optimization to qualify compounds as candidates for structural revalidation.