Feasibility Study of Biosensors Based on Polymelamine‐modified Screen‐printed Carbon Electrodes

We herein report the use of melamine and a low‐cost screen‐printed carbon electrode (SPCE) as the base matrices for the preparation of an electrochemical biosensor. Following the electrochemical polymerization of melamine, the resulting polymelamine was deposited on the SPCE surface to give layers bearing –NH₂ functional groups, which allowed the attachment of anti‐IgE (immunoglobulin E) antibodies. The resulting anti‐IgE‐labeled SPCEs were then incubated with IgE solutions of various concentrations prior to analysis by chronoamperometry using Ru(NH₃)₆³⁺ as an electrochemical mediator. A logarithmic relationship was observed between the chronoamperometric current and the IgE concentration between 5.3 and 530 fM (i. e. over 2 orders of magnitude). In addition, a detection limit of 0.64 fM was achieved in addition to a recovery of 114 ± 14 % for a fetal bovine serum sample spiked with 16 fM IgE. Furthermore, only a small quantity of sample was required for analysis, and the IgE assay was suitable for use in a complex serum matrix without interference. We therefore expect that this novel system will be useful for monitoring the changes in blood IgE levels during the clinical treatment of allergic asthma and rhinitis.     

Electrocatalytic Reduction of Metronidazole on Bismuth Modified Pencil‐lead Electrode

We report a new and simple approach based on an experimental design method for the preparation of pencil‐lead electrode modified with bismuth thin film. The fabrication process consists of reduction of bismuth on the surface of electrode with potentiostate method. Response surface methodology was developed as experimental strategies for modeling and optimization of the influence of some variables on the performance of modified electrode. The electrocatalytic behavior of this modified electrode was exploited as a sensitive detection system for the mercury‐free reduction and determination of metronidazole in pharmaceutical and biological samples by using differential pulse voltammetry and amperometry methods.     

Synthesis and structural characterization of new bioactive ligands and their Bi(III) derivatives

Five new carboxylic acid precursors bearing thiourea group and their corresponding bismuth(III) complexes were synthesized and characterized using CHNS and inductively coupled plasma analyses and infrared and NMR (¹H, ¹³C) spectroscopies. Single‐crystal X‐ray diffraction analysis was also carried out for one of the precursors. The behaviour of the compounds was bioassayed for antibacterial, antifungal, antioxidant and enzyme (lipoxygenase, α‐glycosidase and anti‐urease) inhibition activities. It is concluded that the interaction of the compounds with bismuth enhances both the antimicrobial and enzyme inhibition activities. The synthesized compounds may prove to be good therapeutic agents.     

Comparison of reversed‐phase liquid chromatography and hydrophilic interaction chromatography for the fingerprint analysis of Radix isatidis

Radix isatidis is a famous anti‐influenza virus herbal medicine traditionally taken as a water decoction. However, the chemical fingerprint analysis of Radix isatidis is dominantly based on RPLC, from which it is difficult to obtain fingerprint information of hydrophilic compounds. Here, we developed the separation of Radix isatidis by RPLC and hydrophilic interaction chromatography, comparing the traditional RPLC fingerprint with the hydrophilic interaction chromatography fingerprint. Besides, an anti‐viral assay of Radix isatidis was conducted to evaluate its efficacy. The fingerprint–efficacy relationships between the fingerprints and the anti‐viral activity were further investigated with principal component regression analysis. The results showed that the anti‐viral activity correlated better with the hydrophilic interaction chromatography fingerprint than with the RPLC fingerprint. This study indicates that hydrophilic interaction chromatography could not only be a complementary method to increase the fingerprint coverage of conventional RPLC fingerprint, but also can better represent the efficacy and quality of Radix isatidis.     

Anti‐inflammatory bioactive equivalence of combinatorial components β‐carboline alkaloids identified in Arenaria kansuensis by two‐dimensional chromatography and solid‐phase extraction coupled with liquid–liquid extraction enrichment technology

Bioactive equivalent combinatorial components play a critical role in herbal medicines. However, how to discover and enrich them efficiently is a question for herbal pharmaceuticals researchers. In our work, a novel two‐dimensional reversed‐phase/hydrophilic interaction high‐performance liquid chromatography method was established to perform real‐time components trapping and combining for preparation and isolation of coeluting components. Arenaria kansuensis was taken as an example, and solid‐phase extraction coupled with liquid–liquid extraction as a simple and efficient method for enriching trace components, reversed phase column coupled with hydrophilic interaction liquid chromatography XAmide column as two‐dimensional chromatography technology for isolation and preparation of coeluting constituents, enzyme‐linked immune‐sorbent assay as bio‐guided assay, and anti‐inflammatory bioactivity evaluation for bioactive constituents. A combination of 12 β‐carboline alkaloids was identified as anti‐inflammatory bioactive equivalent combinatorial components from A. kansuensis , which accounts for 1.9% w/w of original A. kansuensis . This work answers the key question of which are real anti‐inflammatory components from A. kansuensis and provides a fast and efficient approach for discovering and enriching trace β‐carboline alkaloids from herbal medicines for the first time. More importantly, the discovery of bioactive equivalent combinatorial components could improve the quality control of herbal products and inspire a herbal medicine based on combinatorial therapeutics.     

Preparation of Nano‐bismuth with Different Particle Sizes and the Size Dependent Electrochemical Thermodynamics

Nano‐bismuth has excellent electrochemical properties. However, it is still unclear how the particle size of nano‐bismuth influences its electrochemical thermodynamic properties. In this paper, spherical bismuth nanoparticles with different particle sizes were prepared by solvothermal method; the electrode potentials, the temperature coefficients of the electrode potentials and the thermodynamic functions of reaction for nano‐bismuth electrodes with different particle sizes at different temperatures were determined; and the effects of particle size on the electrode potential, the temperature coefficient and the thermodynamic functions were discussed. The experimental results show that particle size of bismuth nanoparticles has a significant influences on the electrochemical thermodynamic properties. The standard electrode potential of the nano‐bismuth electrode with a diameter of 39.9 nm was 0.009 V lower than that of the ordinary standard electrode (0.308 V); the temperature coefficient of the electrode potential with a diameter of 39.9 nm was nearly double that of 85.9 nm. With the particle sizes decrease, the standard molar Gibbs energy of reaction, the standard molar enthalpy of reaction, the standard molar entropy of reaction, the molar reversible reaction heat and the temperature coefficient increase; and these quantities are linearly related to the reciprocal of the particle diameter.     

Determination of (4‐(1,3‐dioxo‐1,3‐dihydro‐2H‐isoindol‐ 2‐yl)‐N′‐[(4‐ethoxyphenyl) methylidene] benzohydrazide,a novel anti‐inflammatory agent,in biological fluids by UPLC‐MS/MS: Assay development,validation and in vitro metabolic stability study

A thalidomide analog, (4‐(1,3‐dioxo‐1,3‐dihydro‐2H‐isoindol‐2‐yl)‐N ′‐[(4‐ethoxyphenyl) methylidene] benzohydrazide), has been identified as a promising broad‐spectrum anti‐inflammatory agent in previous study. In this study, a sensitive and selective UPLC‐MS/MS assay was developed and validated for its determination in rat plasma samples. The chromatographic separation was performed on an Aquity BEH C₁₈ column using mobile phase comprising of acetonitrile and 10 mm ammonium acetate in the ratio of 85: 15, at flow rate of 0.3 mL/min. The detection and quantification were performed in positive multiple reaction monitoring mode by parent to daughter ion transition of 414.06 ˃ 148.05 for analyte and 411.18 ˃ 191.07 for internal standard (risperidone), respectively using electrospray ionization source. The sample extraction process consisted of liquid–liquid extraction method using diethyl ether as the extracting solvent. The assay was validated by following FDA guidelines and all parameters were found to be within acceptable limits. The linearity was between 10.1 and 2500 ng/mL and the lower limit of quantification was 10.1 ng/mL. The reported results indicate that the assay could meet the requirement for analysis of this compound in amounts expected to the present in actual samples. Further, in vitro metabolic stability study was performed in rat liver microsomes by using the validated assay.     

Anti‐Clostridium tetani Antibody Determination in Serum Samples by Amperometric Immunosensing

An electrochemical (EC) immunosensing assay for anti‐Clostridium tetani antibody determination in serum has been developed. The antigen tetanus toxoid was immobilized on superparamagnetic nanobeads. The immunoreaction occurred in Eppendorf minitubes. The anti‐tetani antibody was incubated in the presence of the toxoid functionalized nanobeads, then reacted with horseradish peroxidase‐labeled anti‐IgG. The resulting immunobeads were retained onto the carbon paste working electrode with a magnet. Hydroquinone served as redox label. The level of anti‐Clostridium tetani antibody in guinea pig serum samples was determined by amperometry using a carbon based screen‐printed electrode (cSPE) housed onto a magnetic support. The EC response was proportional to the logarithm of the antibody concentration comprised between 0.0046 IU/mL and 0.175 IU/mL with a limit of detection of 0.0046 IU/mL. In order to minimize the matrix effect, the standard addition method was applied. The assay was validated by comparing the EC immunosensing data with those obtained by applying the ELISA method described in the European Pharmacopoeia.     

An Aptamer-Based Competitive Fluorescence Quenching Assay for IgE

A simple competitive fluorescence quenching assay based on aptamer was developed for IgE detection. Two DNA probes were used. One is 5′-end fluorescein-labeled IgE aptamer; the other is 3′-end DABCYL-labeled short DNA, which would hybridize with IgE aptamer to quench the fluorescence. In the presence of IgE, the aptamer-IgE complex formed is strong enough to prevent the short DNA probes hybridizing with the bounded aptamer probes, which results in the less decrease of fluorescence intensity. The signal change was found to be proportional to the concentration of IgE from 0.35 to 35 nM with a detection limit of 0.17 nM.     

Aptamer‐based detection and quantitative analysis of human immunoglobulin E in capillary electrophoresis with chemiluminescence detection

A novel aptamer‐based CE with chemiluminescence (CL) assay was developed for highly sensitive detection of human immunoglobulin E (IgE). The IgE aptamer was conjugated with gold nanoparticles (AuNPs) to form AuNPs‐aptamer that could specifically recognize the IgE to produce an AuNPs‐aptamer‐IgE complex. The mixture of the AuNPs‐aptamer‐IgE complex and the unbounded AuNPs‐aptamer could be effectively separated by CE and sensitively detected with luminol‐H₂O₂ CL system. By taking the advantage of the excellent catalytic behavior of AuNPs on luminol‐H₂O₂ CL system, the ultrasensitive detection of IgE was achieved. The detection limit of IgE is 7.6 fM (S/N = 3) with a linear range from 0.025 to 250 pM. Successful detection of IgE in human serum samples was demonstrated and the recoveries of 94.9–103.2% were obtained. The excellent assay features of the developed approach are its specificity, sensitivity, adaptability, and very small sample consumption. Our design provides a methodology model for determination of rare proteins in biological samples.     

Relationship between the UPLC‐Q‐TOF‐MS fingerprinted constituents from Daphne genkwa and their anti‐inflammatory,anti‐oxidant activities

Daphne genkwa Sieb.et Zucc. is a well‐known medicinal plant. This study was designed to apply the ultra‐high performance liquid chromatography system to establish a quality control method for D. genkwa. Data revealed that there were 15 common peaks in 10 batches of D. genkwa Sieb. Et Zucc. (Thymelaeaceae) from different provinces of China. On this basis, the fingerprint chromatogram was established to provide references for quality control. Afterwards, the chemical constitutions of these common peaks were analyzed using the UPLC‐Q‐TOF‐MS system and nine of them were identified. In addition, LPS‐stimulated RAW264.7 murine macrophages and DPPH assay were used to study the anti‐inflammatory and anti‐oxidation effects of D. genkwa . Then the fingerprint–efficacy relationships between UPLC fingerprints and pharmacodynamic data were studied with canonical correlation analysis. Analysis results indicated that the anti‐inflammatory and anti‐oxidation effects differed among the 10 D. genkwa samples owing to their inherent differences of chemical compositions. Taken together, this research established a fingerprint–efficacy relationship model of D. genkwa plant by combining the UPLC analytic technique and pharmacological research, which provided references for the detection of the principal components of traditional Chinese medicine on bioactivity.     

Screening anti‐allergic components of Astragali Radix using LAD2 cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method

Huangqi (Astragali Radix), a traditional Chinese herb, is widely used in clinical therapy in China. In addition, an anti‐allergic effect of constituents in Huangqi has been reported in the scientific literature. In the present study, cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method was developed to screen, analyze and identify the anti‐allergic components of Huangqi. The Laboratory of Allergic Disease 2 (LAD2) cell was used to establish cell membrane chromatography, which was combined with UHPLC‐ESI‐MS/MS. The coupled system was then used to screen anti‐allergic components from Huangqi. Effects of active components were verified by histamine release assay. A component retained on the LAD2 cell membrane chromatography was identified as formononetin. Bioactivity of formononetin was investigated by histamine release assay in LAD2 cells, and it was found that formononetin could inhibit histamine release in a dose‐dependent manner from 1 to 100 μm . The LAD2 cell membrane chromatography online with UHPLC‐ESI‐MS/MS method is an effective technique for screening the anti‐allergic components of Huangqi.     

Tributyltin(IV)[3‐(3′,5′‐dimethylphenylamido)propanoate] as a potent HCV inhibitor,its delivery study,controlled release and binding sites using CTAB as a standard cell membrane

Gaussia luciferase assay was used to measure the anti‐hepatitis C (anti‐HCV) potency of tributyltin(IV)[3‐(3′,5′‐dimethylphenylamido)propanoate] in infected Huh 7.5 cells (human hepatocellular cell). Interaction of the organotin(IV) complex with cetyl N,N,N‐trimethylammonium bromide (CTAB) micelles was studied using UV–visible and steady‐state florescence spectroscopy. The anti‐HCV study showed a log IC₅₀ value of 0.96 nm for the complex. The complex–CTAB interaction parameter showed that partition of the complex from bulk water to the CTAB micelle was a spontaneous process, and the red shift in visible spectra of the complex confirmed its increased solubility into micelles. Copyright © 2013 John Wiley & Sons, Ltd.     

Pyrido[1,2‐a]pyrimidin‐4‐ones: Ligand‐based Design,Synthesis, and Evaluation as an Anti‐inflammatory Agent

In the present study, a series of novel pyrido[1,2‐a]pyrimidin‐4‐one derivatives ( 1 – 45 ) were synthesized, characterized, and evaluated for their anti‐inflammatory activity. The structures of all newly synthesized compounds were confirmed by ¹H NMR, ¹³C NMR, mass spectroscopy, and C, H, and N analyses. Preliminary these newly synthesized compounds were evaluated for their in vitro cyclooxygenase (COX)‐2/COX‐1 inhibitory activity. The celecoxib, a COX‐2 inhibitor, was used as a reference standard drug. In this inhibitory study, compounds 42 , 43 , 44 , and 45 were found to have significant in vitro inhibitory profile as compared with the reference drug. These compounds were then subjected to their in vivo anti‐inflammatory assay by using carrageenan‐induced rat paw edema method in next level of screening. Later, these same compounds were tested for their ulcerogenic property. Based on these activity data, the compound 43 (in vitro COX‐2 activity—IC₅₀ = 0.4 μM, SI = 400, in vivo anti‐inflammatory activity—72% inhibition after 3 h, and 0.38%—Ulcer index) was emerged as most promising anti‐inflammatory agent with very low ulcerogenic action.     

Determination of DNA Hypermethylation Using Anti‐cancer Drug‐Temozolomide

An electrochemical drug‐DNA biosensor was developed for the detection of interaction between the anti‐cancer drug, Temozolomide (TMZ), and DNA sequences by using Differential Pulse Voltammetry at the graphite electrode surfaces. TMZ is a pro‐drug and an alkylating agent that crosses the blood‐brain barrier, so it is mainly used for brain cancers treatment. In this study, we aim to develop a‐proof‐of‐concept study to investigate the effect of TMZ on formerly methylated DNA sequences since TMZ shows its anti‐cancer activity by methylating the DNA. Interaction between TMZ and DNA causes localized distortion of DNA away from an idealized B‐form, resulting in a wider major groove and greater steric accessibility of functional groups in the base of the groove. According to the results, TMZ behaves as a ‘hybridization indicator’ because of its different electrochemical behavior to different strands of DNA. After interaction with TMZ, hybrid (double stranded DNA‐dsDNA) signals decreased dramatically whereas probe (single stranded DNA‐ssDNA) and control signals remain almost unchanged. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or tag. It is the first study to demonstrate the interaction between the TMZ and dsDNA created from probe and target. We use specific oligonucleotides sequences instead of using long dsDNA sequences.     

Bioactivity‐based ultra‐performance liquid chromatography‐coupled quadrupole time‐of‐flight mass spectrometry for NF‐κB inhibitors identification in Chinese Medicinal Preparation Bufei Granule

Traditional Chinese medicine (TCM) preparations have become effective treatments for many diseases. However, their active ingredients are still uncertain and difficult to identify. In this study, we propose a strategy that integrates ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) and bioactive (NF‐κB inhibitor) luciferase reporter assay systems for the rapid determination of various anti‐inflammatory compounds of TCM preparations. In this way, Bufei Granule (BFG), a TCM preparation used for the clinical therapy of asthma, was analyzed by the two ways of component identification and activity detection. Potential anti‐inflammatory constituents were screened by NF‐κB activity assay systems and simultaneously identified according to the mass spectrometry data. Three structural types of NF‐κB inhibitors (caffeic acid derivatives, flavonoids and Pentacyclic triterpenes) were characterized. Further cytokine detection confirmed the anti‐inflammatory effects of the potential NF‐κB inhibitors. Compared with conventional chromatographic separation and inhibitory activity detection, integrating UPLC/Q‐TOF‐MS identification and virtual validation was more convenient and more reliable. This strategy clearly demonstrates that MS data‐based fingerprinting is a meaningful tool not only in identifying constituents in complex matrix but also in directly screening for powerful trace ingredients in TCM preparations. Copyright © 2016 John Wiley & Sons, Ltd.     

A Three‐dimensional DyIII‐based Metal‐organic Framework: Smart Drug Carrier and Anti‐lung Cancer Activity

A 3D lanthanide metal‐organic framework (MOF) with the formula [Dy₂(L)₂(H₂O)₂]_n ( 1 ) (H₃L = biphenyl‐3,4′,5‐tricarboxylic acid) was synthesized under solvothermal conditions and structurally characterized by elemental analysis, powder X‐ray diffraction analysis, infrared spectroscopy, and single‐crystal X‐ray diffraction analysis. Compound 1 features a 3D porous framework based on 1D rod‐shaped Dy^III‐carboxylate chains. The efficient encapsulation and controllable release of an anticancer drug (5‐Fu) make it a promising drug delivery host. Furthermore, the GCMC simulation was used to probe the drug‐framework interaction at the atomic lever. The in vitro anti‐lung cancer activity of 1 and 5‐Fu loaded 1a were also evaluated using MTT assay.     

Development of an enzyme‐linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti‐glycyrrhizic acid monoclonal antibody

In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti‐glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid–bovine serum albumin conjugate with the hypoxanthine–aminopterin–thymidine‐sensitive mouse myeloma cell line (Sp2/0‐Ag14). Subsequently, an indirect, competitive enzyme‐linked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12–2500 ng/mL. Both intra‐assay and inter‐assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine.     

A Sensitive Electrochemical Immunosensor for α‐Fetoprotein Detection with Colloidal Gold‐Based Dentritical Enzyme Complex Amplification

A sensitive and specific electrochemical immunosensor was developed with α‐fetoprotein (AFP) as the model analyte by using gold nanoparticle label for enzymatic catalytic amplification. A self‐assembled monolayer membrane of mercaptopropionic acid (MPA) was firstly formed on the electrode surface through gold‐sulfur interaction. Monoclonal mouse anti‐human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, gold nanoparticles coated with polyclonal rabbit anti‐human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti‐rabbit IgG conjugated with alkaline phosphatase, the dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the colloidal gold‐based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the concentration of AFP from 1.0 ng mL^?1 to 500 ng mL^?1 with a detection limit of 0.8 ng mL^?1. This system could be extended to detect other target molecules with the corresponding antibody pairs.     

Synthesis and Molecular Drug Efficacy of Indoline‐based Dihydroxy‐thiocarbamides: Inflammation Regulatory Property Unveiled over COX‐2 Inhibition,Molecular Docking,and Cytotoxicity Prospects

In this study, substituted indoline‐based dihydroxy‐carbamides ( 5a–i ) were synthesized and evaluated as the cyclooxygenase‐2 (COX‐2) inhibitors to testify their inflammatory regulations through COX‐2 inhibition. Enzyme‐linked immunosorbent assay‐based competitive (COX‐2) inhibition (in vitro) followed by a molecular docking study (in silico) was executed to ensure the mode of interaction between 5a–i and COX‐2. Apart from COX‐2 inhibition studies, free‐radical scavenging ability (H₂O₂ estimation method) and the human red blood cell membrane protection (in vitro anti‐inflammatory) capability of the compounds 5a–i assessment were also evaluated. Excellent antimicrobial and anticancer activity exhibited by thiocarbamide substituted compounds ( 5a–d ) than carbamide ( 5e–i ). In molecular docking studies, the obtained binding affinity values of 5a–i indicated the therapeutic selectivity on COX‐2 (PDB ID: 1CX2) over COX‐1 (PDB ID: 1EQG). Established inhibitory constant (ki) values were found as low as in nanomolar/picomolar against COX‐2. Reliable COX‐2 inhibition of 78–92% and IC₅₀ 0.002–1.28 μM were obtained. Human red blood cell membrane was found to be effectively stabilized/protected by 5a–i up to 98%. Excellent antioxidant property (average radical scavenging 92%) and structure–activity relationship predictions confirmed the druggability potentials of 5a–i as effective, future anti‐inflammatory drugs. The cytotoxicity of the compounds was also unveiled by MTT assay using MCF‐7 (human breast cancer), SW620 (human colon cancer), G361 (human skin cancer), human breast normal cell lines (MCF‐10), and cell lines.