Benzoic acid-modified monolithic column for separation of hydrophilic compounds by capillary electrochromatography with high content of water in mobile phase.

Hydrophilic column combined with mobile phase containing high content of water is a green method for the separation of polar compounds, but there are few related studies, and the separation efficiency and performance of existing columns still needs to be improved. In this work, a novel monolithic column for separation of hydrophilic compounds under both high water content and HILIC condition, was prepared by in-situ polymerization using 4-vinylbenzoic acid (VBA) and 1-(Acryloyloxy)-3-(methacryloyloxy)-2-propanol (AMAP) as functional monomers. The poly(VBA-co-AMAP) monolithic column showed good separation performance towards various polar compounds under different chromatographic conditions based on the π-interaction, hydrophobic and hydrogen bonding interactions provided by 4-vinylbenzoic acid functional monomer. The highest column efficiency for adenine was over 2.15 × 105 plates m-1 (theoretical plate, N). In addition, the monolith showed good stability and reproducibility, the relative standard deviations (RSDs) of retention times within days (n = 5), between days (n = 5), between columns (n = 3) and between batches (n = 3) were 0.47-1.13%, 1.20-2.68%, 0.59-1.78% and 1.54-3.60%, respectively. This novel type of monolith has great application potential in the separation of hydrophilic compounds.

Novel strategy of electrochemical analysis of DNA bases with enhanced performance based on copper-nickel nanosphere decorated N,B-doped reduced graphene oxide.

Design of suitable nanocomposites with tailored structures was significant in the fabrication of effective and reliable electrochemical sensors. Herein, the copper-nickel@nitrogen, boron-doped reduced graphene oxide (Cu-Ni@N,B-rGO) was successfully synthesized, which exhibited superior electrocatalytic performance towards guanine (G) and adenine (A) oxidation. The Cu-Ni NPs were sequentially decorated on N,B-rGO substrate via an environmentally friendly reduction strategy, which utilized glucose as reducer and stabilizing agent. The nanocomposites with large specific surface area, remarkable conductivity and high catalytic activity showed prominent synergistic effect owning to the uniform dispersion of Cu-Ni NPs on the surface of N,B-rGO. When applied to analysis of G and A using DPV, the wide linear ranges of 1.0-160.0 µM and 1.0-120.0 µM with the determination limits of 0.118 µM and 0.134 µM were obtained, respectively. The sensor was successfully applied to the detection of G and A in calf-thymus DNA with G/A ratio of 0.80. The facile preparation process and attractive sensing properties of the Cu-Ni@N,B-rGO nanocomposites made it a promising candidate for the development of advanced electrochemical sensor.

Plasmonic-3D photonic crystals microchip for surface enhanced Raman spectroscopy.

Plasmonic-dielectic hybrid substrates of Ag-islands on three-dimensional photonic crystals are fabricated through magnetron sputtering of silver onto hydrophobized silica photonic crystals, free from etching process. Without typical "hot-spots" such as nanogaps, significant Raman enhancements can be achieved, attributed to the enhanced electromagnetic field and scattering of the plasmonic nanoparticles as well as the enhanced light-matter interaction by the slow photon effects. The detection limit for adenine by the hybrid substrates reaches nM level, with a calculated enhancement factor of 1.13 × 107, which is three orders of magnitude higher than the conventional noble metal film over nanosphere (FON) control group. Furthermore, microchips based on the hybrid substrates are facilely achieved, enabling micro-detection through super hydrophobic concentration. The facile fabrication and effective Raman enhancements make the Ag-islands on 3D photonic crystals promising candidates in the field of chemical sensors, Raman mapping and bioassays.

Photoelectrochemical immunosensor for N6-methyladenine detection based on Ru@UiO-66, Bi2O3 and Black TiO2.

A novel photoelectrochemical (PEC) immunosensor was successfully constructed for N6-methyladenine (m6A) detection based on the photoactive materials of black titanium dioxide (B-TiO2) and bismuth trioxide (Bi2O3) and the signal amplification unit of [Ru(bpy)3]2+-doped metal organic framework (MOF). The Bi2O3/B-TiO2/ITO electrode was first fabricated, then decorated with gold nanoparticles (AuNPs) which provided sites for anchoring m6A antibodies. After the capture of m6A via immunoreaction with the antibody, the Zr-based metal organic framework (UiO-66)-[Ru(bpy)3]2+ compound was further attached specifically to the phosphate group of m6A. With visible light irradiation, a large and stable photocurrent response was produced in the presence of ascorbic acid (AA). Under optimized experimental conditions, the linear range of the PEC biosensor was 0.05-30 nM, with a low detection limit of 0.0167 nM (S/N = 3). This method showed high specificity, selectivity, stabilization and repeatability. Moreover, it was successfully used for the detection of m6A in rice seedling leaves that had been subjected to heavy metal treatment during their development.

New adenine analogues and a pyrrole alkaloid from Selaginella delicatula.

Phytochemical study on the n-BuOH extract of Selaginella delicatula lead to the isolation, characterization and structure elucidation of two new adenine analogues, delicatulines A (1) and B (2), one new pyrrole alkaloid (4), and five known compounds (3, 5-8). These new substances all contain an aliphatic chain in their parent nucleus, which were unusual to find in plants. In the present study, they were identified from Selaginellaceae for the first time. The structures and absolute configurations of these new compounds were determined by a combination of NMR and CD spectroscopic analyses. Compounds 1, 3 and 4 were evaluated for their inhibitory activities on HBV surface antigen and HBV DNA in HepAD38 cells. The results showed that these compounds had only weak or no inhibitive effects on HBV.

Single step grown MoS2 on pencil graphite as an electrochemical sensor for guanine and adenine: A novel and low cost electrode for DNA studies.

Herein we report a simple, one-step approach to prepare a low-cost and binder free MoS2-pencil graphite electrode (i.e., MoS2-PGE) for the electrochemical oxidation of DNA nucleobases i.e., guanine (G) and adenine (A) in physiological pH (7.4) buffer solution. MoS2-PGE was synthesised by hydrothermal method and the morphology of such hybrid was characterized by field emission scanning electron microscopy, X-ray diffraction, Raman and X-ray photoelectron spectroscopy. In cyclic voltammetry, MoS2-PGE displays two well-seprated and well-defined irresversible peaks at 0.58 and 0.90 V for electrochemical oxidation of G and A respectively when compared to bare PGE. Likewise, differential pulse voltammetry of MoS2-PGE showed well-seprated and sharp peak current responses for G and A at 0.56 V and 0.85 V respectively. Under optimized conditions, DPV was further adopted for simultaneous and separation-free determination of G and A in physiological pH. MoS2-PGE shows good stability with linear range of 15-120 µM and 15-120 µM for G and A detection respectively. Obtained sensitivity and limit of detection (signal-to-noise = 3) are comparable with the previous literature. As an immediate practical applicability, MoS2-PGE was used for quantification of G and A concentration in calf-thymus DNA and detected ratio of G and A (i.e., [G]/[A]) ratio is 0.85. The current approach provides a new avenue towards the development of affordable electrodes for a wide range of bioanalytical applications.

Chemical constituents from Gnaphalium affine and their xanthine oxidase inhibitory activity.

Gnaphalium affine D. Don, a medicinal and edible plant, has been used to treat gout in traditional Chinese medicine and popularly consumed in China for a long time. A detailed phytochemical investigation on the aerial part of G. affine led to the isolation of two new esters of caffeoylquinic acid named (-) ethyl 1, 4-di-O-caffeoylquinate (1) and (-) methyl 1, 4-di-O-caffeoylquinate (2), together with 35 known compounds (3-37). Their structures were elucidated by spectroscopic data and first-order multiplet analysis. All the isolated compounds were tested for their xanthine oxidase inhibitory activity with an in vitro enzyme inhibitory screening assay. Among the tested compounds, 1 (IC50 11.94 µmol·L-1) and 2 (IC50 15.04 µmol·L-1) showed a good inhibitory activity. The current results supported the medical use of the plant.

Graphene oxide reinforced core-shell structured Ag@Cu2O with tunable hierarchical morphologies and their morphology-dependent electrocatalytic properties for bio-sensing applications.

In this study, a facile solution approach was developed for the synthesis of a series of core-shell structured Ag@Cu2O nanocrystals of various shapes including triangles, spheres, and cubes with well-defined stable heterojunctions. The electrooxidation of dopamine (DA), uric acid (UA), guanine (G), and adenine (A) using these hybrids revealed morphology-dependent sensing properties, with activities and accumulation ability following the order, triangular Ag@Cu2O > spherical Ag@Cu2O > cubic Ag@Cu2O. Further, we constructed a novel graphene oxide (GO) nanosheet-reinforced triangular Ag@Cu2O ternary hetero-nanostructure. Such a hybrid with a three-dimensional interconnected hierarchical architecture is suitable for catalysis, since it not only leads to improved interfacial electron transfer, but also readily exposes the highly catalytic Ag@Cu2O to the reactants. Therefore, more enhanced electrochemical activities were observed for the oxidation of DA, UA, G, and A. This study provides an efficient way to synthesize morphology-controlled Ag@Cu2O heterogeneous catalysts for the fabrication of potential biosensors, and also opens up attractive avenues in the design of multifunctional ternary noble metal-semiconductor-carbon hybrids.

Tomenphantadenine, an unprecedented germacranolide-adenine hybrid heterodimer from the medicinal plant Elephantopus tomentosus L.

An unusual adenine-substituted germacrane sesquiterpene lactone, tomenphantadenine (1), has been isolated from the whole plant of Elephantopus tomentosus L. The structure of this compound was established by comprehensive spectroscopic analysis including high resolution (HR) ESI-MS, 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data. This compound features novel hybrid pattern of germacrane sesquiterpene with adenine through C-N linkage, and a possible biosynthetic pathway for it was proposed. Compound 1 showed potent antibacterial activity against the gram-positive Staphylococcus aureus and weak acetylcholinesterase (AChE) inhibitory activity.

A novel electrochemical sensor based on Cu@Ni/MWCNTs nanocomposite for simultaneous determination of guanine and adenine.

A novel electrochemical sensing platform based on combination of multi-walled carbon nanotubes and copper-nickel hybrid nanoparticles (Cu@Ni/MWCNTs) was developed for simultaneous detection of guanine (G) and adenine (A). The Ni/MWCNTs and Cu@Ni/MWCNTs nanocomposites were characterized by transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS). The electrochemical behaviors of G and A on the modified electrode were explored by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in phosphate buffer with pH 3.0. Under the optimal conditions, electrical signals were linear over the concentration ranges from 5.0 to 180µM and 8.0 to 150µM for simultaneous determination G and A with the detection limit as low as 0.35µM and 0.56µM (S/N = 3), respectively. Furthermore, linear concentration ranges in individual determination are 1.0-180µM and 2.0-150µM with detection limits of 0.17µM and 0.33µM (S/N = 3) for G and A, respectively. The sensor was successfully used to quantify G and A in real samples. The Cu@Ni/MWCNTs composite presented here can serve as a promising candidate for developing electrochemical sensor devices and plays an important role in widespread fields.

Determination of the Main Nucleosides and Nucleobases in Natural and Cultured Ophiocordyceps xuefengensis.

Ophiocordyceps xuefengensis, a recently described species of Ophiocordycepsthat is associated with the larvae of Phassusnodus (Hepialidae) in the living root or trunk of the medicinal plant Clerodendrumcyrtophyllum, isthe largest known Cordycepsspecies and is recognized as a desirable alternative for natural Ophiocordycepssinensis. This study investigated the main nucleosides and nucleobases in natural and cultured Ophiocordycepsxuefengensis. The contents of the nucleosides and nucleobases in the natural and cultured samples were determined by reverse phase HPLC. The highest concentration of adenosine was found in the natural fruit body and the cultured stroma, with almost no adenosine in the cadaver of Phassusnodus. The contents of adenine, guanosine, uridine and uracil in the cultured mycelium were significantly higher than those in the natural sample. Inosine was only detected in the natural samples. Thymidine and 2-deoxyadenosine were only found in the cadaver of Phassusnodus. Cordycepin was not detected in the five samples examined. These results suggested that the cultured mycelium and cultured stroma of Ophiocordycepsxuefengensis might be a promising substitute for natural O. xuefengensis.

A novel pretreatment method combining sealing technique with direct injection technique applied for improving biosafety.

AIM: People today have a stronger interest in the risk of biosafety in clinical bioanalysis. A safe, simple, effective method of preparation is needed urgently. METHODOLOGY/RESULTS: To improve biosafety of clinical analysis, we used antiviral drugs of adefovir and tenofovir as model drugs and developed a safe pretreatment method combining sealing technique with direct injection technique. The inter- and intraday precision (RSD %) of the method were <4%, and the extraction recoveries ranged from 99.4 to 100.7%. Meanwhile, the results showed that standard solution could be used to prepare calibration curve instead of spiking plasma, acquiring more accuracy result. CONCLUSION/DISCUSSION: Compared with traditional methods, the novel method not only improved biosecurity of the pretreatment method significantly, but also achieved several advantages including higher precision, favorable sensitivity and satisfactory recovery. With these highly practical and desirable characteristics, the novel method may become a feasible platform in bioanalysis.

Sequence-dependent separation of trinucleotides by ion-interaction reversed-phase liquid chromatography-A structure-retention study assisted by soft-modelling and molecular dynamics.

We studied sequence-dependent retention properties of synthetic 5'-terminal phosphate absent trinucleotides containing adenine, guanine and thymine through reversed-phase liquid chromatography (RPLC) and QSRR modelling. We investigated the influence of separation conditions, namely mobile phase composition (ion interaction agent content, pH and organic constituent content), on sequence-dependent separation by means of ion-interaction RPLC (II-RPLC) using two types of models: experimental design-artificial neural networks (ED-ANN), and linear regression based on molecular dynamics data. The aim was to determine those properties of the above-mentioned analytes responsible for the retention dependence of the sequence. Our results show that there is a deterministic relation between sequence and II-RPLC retention properties of the studied trinucleotides. Further, we can conclude that the higher the content of ion-interaction agent in the mobile phase, the more prominent these properties are. We also show that if we approximate the polar component of solvation energy in QSRR by the electrostatic work in transferring molecules from vacuum to water, and the non-polar component by the solvent accessible surface area, these parameters best describe the retention properties of trinucleotides. There are some exceptions to this finding, namely sequences 5'-NAN-3', 5'-ANN-3', 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' (N stands for generic nucleotide). Their role is still unknown, but since linear regression including these specific constellations showed a higher observable variance coverage than the model with only the basic descriptors, we may assume that solvent-analyte interactions are responsible for the exceptional behaviour of 5'-NAN-3' & 5'-ANN-3' trinucleotides and some intramolecular interactions of neighbouring nucleobases for 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' trinucleotides.

Adeninealkylresorcinol, the first alkylresorcinol tethered with nucleobase from Lasiodiplodia sp.

Adeninealkylresorcinol (1), an unusual alkylresorcinol with adenine-alkylresorcinol conjoined skeleton, was isolated from an endophytic fungus Lasiodiplodia sp. obtained from a traditional Chinese medicine Houttuynia cordata Thunb., together with three new biogenetically related compounds (2-4). Their structures were elucidated by comprehensive spectroscopic analysis, and the absolute configuration of 4 was determined by the modified Mosher's method and quantum chemical calculation. Among them, adeninealkylresorcinol (1) is the first alkylresorcinol tethered with nucleobase. In addition, the antioxidant, cytotoxic, and antimicrobial activities of 1-3 were evaluated.

Eritadenine from Edible Mushrooms Inhibits Activity of Angiotensin Converting Enzyme in Vitro.

The inhibition of angiotensin converting enzyme (ACE) activity was determined in vitro by mushroom-derived eritadenine (EA), which was analyzed in 11 principal Korean edible mushrooms. EA inhibited ACE activity with 0.091 µM IC50, whereas the IC50 of captopril (CP), which is a reference compound, was 0.025 µM. Kinetic measurements of ACE reaction in the substrate of hippuryl-l-histidyl-l-leucine (HHL) with or without EA revealed that the Vmax (0.0465 O.D/30 min) was unchanged, but the the Km increased from 2.063 to 3.887 mM, indicating that EA competes with HHL for the active site. When EA was analyzed by HPLC, Lentinus edodes with a soft cap contained the highest amount EA (642.8 mg%); however, Phellinus linteus with a hard cap contained the least amount of EA (9.4 mg%). These results indicate that EA was a strong competitive inhibitor for ACE, and edible mushrooms with soft caps contained a significant amount of EA.

Water-compatible molecularly imprinted polymer as a sorbent for the selective extraction and purification of adefovir from human serum and urine.

A molecularly imprinted polymer has been synthesized to specifically extract adefovir, an antiviral drug, from serum and urine by dispersive solid-phase extraction before high-performance liquid chromatography with UV analysis. The imprinted polymers were prepared by bulk polymerization by a noncovalent imprinting method that involved the use of adefovir (template molecule) and functional monomer (methacrylic acid) complex prior to polymerization, ethylene glycol dimethacrylate as cross-linker, and chloroform as porogen. Molecular recognition properties, binding capacity, and selectivity of the molecularly imprinted polymers were evaluated and the results show that the obtained polymers have high specific retention and enrichment for adefovir in aqueous medium. The new imprinted polymer was utilized as a molecular sorbent for the separation of adefovir from human serum and urine. The serum and urine extraction of adefovir by the molecularly imprinted polymer followed by high-performance liquid chromatography showed a linear calibration curve in the range of 20-100 µg/L with excellent precisions (2.5 and 2.8% for 50 µg/L), respectively. The limit of detection and limit of quantization were determined in serum (7.62 and 15.1 µg/L), and urine (5.45 and 16 µg/L). The recoveries for serum and urine samples were found to be 88.2-93.5 and 84.3-90.2%, respectively.

Bioactive metabolites isolated from Penicillium sp. YY-20, the endophytic fungus from Ginkgo biloba.

Six known metabolites, adenosine (1), methyl ß-D-ribofuranoside (2), adenine (3), 2'-deoxyadenosine (4), 3-methylpiperazine-2,5-dione (5) and 2'-deoxyuridine (6), were isolated from the extracts of the endophytic fungus Penicillium sp. YY-20 isolated from the root of Ginkgo biloba, and their structures were elucidated by spectroscopic methods. The antioxidant and growth-promoting activities of these compounds were first evaluated. The results indicated that compounds 1, 3 and 4 exhibited potential DPPH-scavenging activities compared with positive control. In addition, all the compounds (except 5) stimulated seed germination of Raphanus sativus, Brassica napus and Brassica chinensis but had weak stimulating effect on their root and hypocotyl growth.

Identification and analysis of the constituents in an aqueous extract of Tricholoma matsutake by HPLC coupled with diode array detection/electrospray ionization mass spectrometry.

The main constituents in an aqueous extract of Tricholoma matsutake (Tm) were identified by high-performance liquid chromatography coupled with diode array detection and electrospray ionization time-of-flight mass spectrometry (HPLC-DAD/TOF-MS) and ion trap mass spectrometry (HPLC-DAD/Trap-MSn). The main factors in the extraction process which affect the yields of nutrients were optimized by single-factor experiments and orthogonal experiment design. In total, 12 constituents were identified from the aqueous extract of Tm, including tyrosine, cytidine, uridine, eritadenine, phenylalanine, nicotinamide, inosine, guanosine, tryptophan, adenosine, 5'-deoxy-5'-methylthioadenosine and riboflavin. The optimized extraction conditions were: the ratio of water to sample was 10 : 1 (v/w), Tm was extracted by ultrasonic-assisted extraction for 10 min, followed by water bath heating at 60 °C for 1 h. Among these extraction factors, the heating temperature is significant based on analysis of variance (ANOVA). The yields of nutrients were affected dramatically at high temperature leading to the loss of nutrients, especially for nucleosides and some amino acids.

Optimization of global DNA methylation measurement by LC-MS/MS and its application in lung cancer patients.

Global analyses of DNA methylation contribute important insights into biology and the wide-ranging role of DNA methylation. We describe the use of online solid-phase extraction and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of 5-methyl-2'-deoxycytidine (5-medC) and 2'-deoxycytidine (dC) in DNA. With the incorporation of isotope internal standards and online enrichment techniques, the detection limit of this method was estimated to be as low as 0.065 pg which enables human global DNA methylation detection using only picogram amounts of DNA. This method was applied to assess the optimal amounts of enzymes required for DNA digestion regarding an accurate global DNA methylation determination and completeness of digestion and to determine global methylation in human tumor adjacent lung tissue of 79 lung cancer patients. We further determined methylated (N7-methylguanine (N7-meG), O (6)-methylguanine (O (6)-meG), and N3-methyladenine (N3-meA)) and oxidized DNA lesions (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)) in lung cancer patients by LC-MS/MS. Optimization experiments revealed that dC was liberated from DNA much more readily than 5-medC by nuclease P1 and alkaline phosphatase (AP) in DNA, which could lead to an error in the global DNA methylation measurement following digestion with insufficient enzymes. Nuclease P1 showed more differential activity for 5-medC and dC than AP. Global DNA methylation levels in adenocarcinoma and squamous cell carcinoma patients were similar in the range of 3.16-4.01 %. Global DNA methylation levels were not affected by smoking and gender and were not correlated with N7-meG or 8-oxodG in lung cancer patients. Levels of O (6)-meG and N3-meA were however found to be undetectable in all lung tissue samples.

Direct-acting DNA alkylating agents present in aqueous extracts of areca nut and its products.

Areca nut is a carcinogen to humans and has been strongly associated with oral premalignant and malignant diseases. Previous studies speculated the presence of unknown direct-acting mutagens present in aqueous extracts of areca nut. We hypothesized whether any direct-acting alkylating agents are present in areca nut and its commercial products. In this study, calf thymus DNA was treated with four different aqueous extracts obtained from unripe and ripe areca nuts or their commercial products, namely, pan masala (without tobacco) and gutkha (with tobacco). Three N-alkylated purines including N7-methylguanine (N7-MeG), N3-methyladenine (N3-MeA), and N7-ethylguanine (N7-EtG) were detected using sensitive and specific isotope-dilution liquid chromatography-tandem-mass spectrometry (LC-MS/MS) methods. The results showed that four types of aqueous extracts significantly induced the formation of N7-MeG and N3-MeA in a linear dose-response manner. Extracts from unripe areca nut exhibited higher methylating potency than those of ripe areca nut, while gutkha had higher methylating potency than pan masala. Meanwhile, gutkha made with areca nut and tobacco, was the only extract found to induce the formation of N7-EtG. Overall, this study first demonstrated that the presence of direct-acting alkylating agents in areca nut and its commercial products exist at a level that is able to cause significant DNA damage. Our findings may provide another mechanistic rationale for areca nut-mediated oral carcinogenesis and also highlight the importance and necessity of the identification of these direct-acting alkylating agents.