Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma.

Novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were employed as the support for high performance affinity chromatography. Heparin was covalently attached to PGMA beads by three different coupling methods. Heparin-PGMA-I was prepared by directly coupling amino-groups of heparin with PGMA. Heparin-PGMA-II and III were prepared by the coupling of heparin to amino-PGMA, which was obtained by amination of PGMA. Heparin-PGMA-II was prepared by coupling the carboxyl groups of heparin to amino-PGMA by using water-soluble carbodiimide as coupling reagent, and heparin-PGMA-III was prepared by the reductive amination of heparin and amino-PGMA with sodium cyanoborohydride. The heparin contents of heparin-PGMA-I, II and III were 1.6, 10.2 and 1.0 mg/g beads, respectively. Their affinity capacities for antithrombin III were investigated. Their binding activity to antithrombin III was not proportional to the content of heparin immobilized, and heparin-PGMA-I was the most efficient affinity medium for antithrombin III. The resultant affinity media presented minimal non-specific interaction with other proteins and can be used in a wide pH range. All the three heparin-PGMA beads were exploited for the separation of antithrombin III from human plasma. The purity of antithrombin III obtained was higher than 90%, which was confirmed by high performance size exclusion chromatography.     

Direct electrochemistry of glucose oxidase based on its direct immobilization on carbon ionic liquid electrode and glucose sensing

Direct electrochemistry of glucose oxidase (GOx) has been achieved by its direct immobilization on carbon ionic liquid electrode (CILE) with a conductive hydrophobic ionic liquid, 1-butyl pyridinium hexafluophosphate ([BuPy][PF₆]) as binder for the first time. A pair of reversible peaks is exhibited on GOx/CILE by cyclic voltammetry. The peak-to-peak potential separation (ΔE_P) of immobilized GOx is 0.056 V in 0.067 M phosphate buffer solution (pH 6.98) with scan rate of 0.1 V/s. The average surface coverage and the apparent Michaelis–Menten constant are 6.69 × 10⁻¹¹ mol·cm⁻² and 2.47 μM. GOx/CILE shows excellent electrocatalytic activity towards glucose determination in the range of 0.1–800 μM with detection limit of 0.03 μM (S/N = 3). The biosensor has been successfully applied to the determination of glucose in human plasma with the average recoveries between 95.0% and 102.5% for three times determination. The direct electrochemistry of GOx on CILE is achieved without the help of any supporting film or any electron mediator. GOx/CILE is inexpensive, stable, repeatable and easy to be fabricated.     

A new type of capillary column for open-tubular electrochromatography

A new type of open-tubular C(18) ester-bonded electrochromatographic column was prepared with sol-gel technology, followed by an on-column octadecyl silylation reaction. Glycidoxypropyltrimethoxysilane, a widely used and important silane agent, was used as the sol-gel precursor to form a thin coating layer on the wall of the fused-silica capillary. The C(18) groups were introduced into the coating layer by on-column esterification reaction with stearic acid. The electrochromatography behavior of the column was evaluated in terms of the separation of peptides. A high efficiency of 4.8x10(5) plates/m was achieved for a basic pentapeptide using the C(18 )ester-bonded column. In comparison with bare capillaries and glycidoxypropyltrimethoxysilane sol-gel-coated capillaries, the C(18) ester-bonded column showed the best separation of a mixture of seven pentapeptides under identical conditions of buffer, pH, and applied voltage.     

Ex vivo identification of circulating tumor cells in peripheral blood by fluorometric “turn on” aptamer nanoparticles

The detection of the circulating tumor cells (CTCs) detached from solid tumors has emerged as a burgeoning topic for cancer diagnosis and treatment. The conventional CTC enrichment and identification mainly rely on the specific binding of the antibodies on the capture interface of the magnetic nanoparticles with the corresponding biomarkers on the cell membranes. However, these methods could easily generate false-negative results due to the extremely low concentration of CTCs and the internal heterogeneity of the tumor cells. Herein, with the aim of selectively identifying CTCs and improving the detection accuracy in peripheral blood, we designed the fluorometric “turn on” Au nanoparticles (DHANs) with the modification of a tumor-targeted moiety, dehydroascorbic acid (DHA) and a fluorometric aptamer, which could be “switched-on” by an over-expressed intracellular protein, namely hypoxia-inducible factor-1α (HIF 1α). This novel nanoformulated detection platform demonstrated the great capacity for visualizing various CTCs in peripheral blood with significantly improved detection efficiency and sensitivity. As a result, the nanoplatform has a great potential to be further applied for CTC detection in vitro or in vivo, which holds promise for extensive CTC studies.

The detection of the circulating tumor cells (CTCs) detached from solid tumors has emerged as a burgeoning topic for cancer diagnosis and treatment.     

改进遗传算法用于未知组分数的重叠色谱峰的解析

按照指数修正的Gauss卷积色谱峰模型，构造了结合模拟退火及变长染色体的改进遗传算法，并结合爬山法，可以方便快捷地进行数目未知的重叠色谱峰的精确解析；此法成功地应用于大鼠脑微透析液氨基酸定量分析中，解析结果良好。     

A dual 13C and 15N long-term labelling technique to investigate uptake and translocation of C and N in beech (Fagus sylvatica L.)

A continuous dual 13CO2 and 15NH4(15)NO3 labelling experimental set-up is presented that was used to investigate the C and N uptake and allocation within 3-year old beech (Fagus sylvatica L.) during one growing season. The C and N allocation pattern was determined after six, twelve and eighteen weeks of growth. The carbon uptake was distinctly different in the three phases examined: The first six weeks after budbreak were dedicated to leaf growth with a R/S (root to shoot) ratio of 0.14 for the new carbon. The second growth phase showed a balanced R/S ratio of C allocation and after week 13, the root compartment was the main carbon sink (R/S = 6.97). Nitrogen allocation was more basipetal as compared to carbon. In the second growth phase, R/S of Nnew was 5.57 but fell to 3.54 for the third growth phase probably due to formation of reserves in buds and stem.     

Activity Enhancement of G‐Quadruplex/Hemin DNAzyme by Flanking d(CCC)

G‐quadruplex (G4)/hemin DNAzymes have been extensively applied in bioanalysis and molecular devices. However, their catalytic activity is still much lower than that of proteinous enzymes. The G4/hemin DNAzyme activity is correlated with the G4 conformations and the solution conditions. However, little is known about the effect of the flanking sequences on the activity, though they are important parts of G4s. Here, we report sequences containing d(CCC), flanked on both ends of the G4‐core sequences remarkably enhance their DNAzyme activity. By using circular dichroism and UV‐visible spectroscopy, the d(CCC) flanking sequences were demonstrated to improve the hemin binding affinity to G4s instead of increasing the parallel G4 formation, which might explain the enhanced DNAzyme activity. Meanwhile, the increased hemin binding ability promoted the degradation of hemin within the DNAzyme by H₂O₂. Furthermore, the DNAzyme with d(CCC) flanking sequences showed strong tolerance to pH value changes, which makes it more suitable for applications requiring wide pH conditions. The results highlight the influence of the flanking sequences on the DNAzyme activity and provide insightful information for the design of highly active DNAzymes.     

Characterization and application of a DNA aptamer binding to L-tryptophan

DNA aptamers for specific recognition of L-tryptophan have been evolved by a SELEX (systematic evolution of ligands by exponential enrichment) technique. Truncation-mutation experiments suggest that a 34-mer sequence, Trp3a-1, possesses the strongest binding ability to L-tryptophan. Trp3a-1 is predicted to adopt a loop-stem secondary structure, in which the loop may further fold into a binding pocket for L-tryptophan with the help of the stem. The specificity investigation shows that Trp3a-1 strongly binds to L-tryptophan, has almost no binding to other amino acids, and weakly binds to some tryptophan analogs and peptides containing the L-tryptophan residue. The binding of Trp3a-1 to L-tryptophan is mainly contributed to by hydrogen bonds and precise stacking formed between the binding pocket of Trp3a-1 and all groups on L-tryptophan. This aptamer has also been proved to be an effective ligand for the chiral separation of D/L-tryptophan. L-tryptophan and its derivatives are known to play important biological roles; this aptamer ligand could be used as a tool for the analysis of tryptophan and other related studies.     

The study of thermal decomposition kinetics of zinc oxide formation from zinc oxalate dihydrate

This study is devoted to the thermal decomposition of ZnC₂O₄·2H₂O, which was synthesized by solid-state reaction using C₂H₂O₄·2H₂O and Zn(CH₃COO)₂·2H₂O as raw materials. The initial samples and the final solid thermal decomposition products were characterized by Fourier transform infrared and X-ray diffraction. The particle size of the products was observed by transmission electron microscopy. The thermal decomposition behavior was investigated by thermogravimetry, derivative thermogravimetric and differential thermal analysis. Experimental results show that the thermal decomposition reaction includes two stages: dehydration and decomposition, with nanostructured ZnO as the final solid product. The Ozawa integral method along with Coats–Redfern integral method was used to determine the kinetic model and kinetic parameters of the second thermal decomposition stage of ZnC₂O₄·2H₂O. After calculation and comparison, the decomposition conforms to the nucleation and growth model and the physical interpretation is summarized. The activation energy and the kinetic mechanism function are determined to be 119.7 kJ mol^?1 and G(α) = ?ln(1 – α)^1/2, respectively.     

A label-free electrochemical biosensor based on a DNA aptamer against codeine

In order to develop a sensor for opium alkaloid codeine detection, DNA aptamers against codeine were generated by SELEX (systematic evolution of ligands by exponential enrichment) technique. An aptamer HL7-14, which is a 37-mer sequence with K_d values of 0.91 μM, was optimized by the truncation-mutation assay. The specificity investigation shows that HL7-14 exhibits high specificity to codeine over morphine, and almost cannot bind to other small molecule. With this new selected aptamer, a novel electrochemical label-free codeine aptamer biosensor based on Au-mesoporous silica nanoparticles (Au-MSN) as immobilized substrate has been proposed using [Fe(CN)₆]^3−/4− as electroactive redox probe. The linear range covered from 10 pM to 100 nM with correlation coefficient of 0.9979 and the detection limit was 3 pM. Our study demonstrates that the biosensor has good specificity, stability and well regeneration. It can be used to detect codeine.