Improved sample preparation method for glycan analysis of glycoproteins by CE‐LIF and CE‐MS

CE is a high‐resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37°C, using ≥100 fold excess of fluorophore reagent, 8‐aminopyrene‐1,3,6‐trisulfonic‐acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:≥100 ratio) maintained the >95% derivatization yield at 55°C with only 50 min reaction time. Terminal sialic acid loss was negligible at 55°C during the derivatization process, and indicating that the kinetics of labeling at 55°C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8‐aminopyrene‐1,3,6‐trisulfonic‐acid simplified the removal of excess reagent, important in both CE‐LIF (electrokinetic injection bias) and CE‐MS (ion suppression). Coupling CE‐ ESI‐MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values.     

Stable second‐order NLO semi‐IPN system based on bipyridine‐containing polyimide and alkoxysilane dye

An organosoluble polyimide based on bipyridyl moiety and an alkoxysilane dye have been developed for second‐order non‐linear optics (NLOs). This bipyridine‐containing polyimide exhibits a glass transition temperature of 254°C and a degradation temperature of 400°C. An NLO‐active semi‐interpenetrating network (IPN) system was prepared by blending the polyimide with the alkoxysilane dye via in situ sol‐gel process of alkoxysilane. The selection of this bipyridine‐containing polyimide as the polymeric matrices provides improved solubility and thermal stability, and most importantly enhanced intermolecular interactions. No aggregation of the NLO chromophores in the polyimide matrices was observed through morphology and NLO studies. Under the limitation of chromophore degradation at elevated temperatures, the pristine poled/cured alkoxysilane dye exhibits poorer long‐term stability. By introducing the polyimide upon a silica network by the semi‐IPN system, randomization of the oriented chromophores can be effectively suppressed. Using in situ contact poling, the r₃₃ coefficients of 2.2–17.0 pm/V were obtained for the optically clear semi‐IPN NLO materials. Excellent temporal stability (100°C) was also achieved for these semi‐IPN materials. Copyright © 2005 John Wiley & Sons, Ltd.     

Focusing and stabilization of bis‐intercalating dye–DNA complexes for high‐sensitive CE‐LIF DNA analysis

Multiple labeling of nucleic acids by intercalative dyes is a promising method for ultrasensitive nucleic acid assays. The properties of the fast dissociation and instability of dye–DNA complexes may prevent from their wide applications in CE‐LIF nucleic acid analysis. Here, we describe an optimum CE focusing method by using appropriately paired sample and separation buffers, Tris‐glycine buffer and Tris‐glycine‐acetic acid buffer. The developed method was applied in both uncoated and polyacrylamide coated fused‐silica capillary‐based CE‐LIF analysis while the sample and separation buffers were conversely used. The complexes of intercalative dye benzoxazolium‐4‐pyridinium dimer and dsDNA were greatly focused (separation efficiency: 1.8 million theoretical plates per meter) by transient isotachophoresis mechanism in uncoated capillary, and moderately focused by transient isotachophoresis in combination of field amplified sample stacking and further stabilized by the paired buffer in polyacrylamide coated capillary. Based on the developed focusing strategy, an ultrasensitive DNA assay was developed for quantitation of calf thymus dsDNA (from 0.02 to 2.14 pM). By the use of an excitation laser power as low as 1 mW, the detection limits of calf thymus dsDNA (3.5 kb) are 7.9 fM in concentration and 2.4×10^?22 mol (150 molecules) in mass. We further demonstrate that the non‐gel sieving CE‐LIF analysis of DNA fragments can be enhanced by the same strategy. Since the presented strategy can be applied to uncoated and coated capillaries and does not require special device, it is also reasonable to extend to the applications in chip‐based CE DNA analysis.     

Thermal degradation and pyrolysis study of brominated butyl rubber‐based damping material

This paper deals with the thermal stability and decomposition behavior of brominated butyl rubber‐based damping material (BRP). The raw materials, butyl rubber matrix (IIR) and brominated phenolic resin (PF), were also investigated as control. IIR shows one decomposition stage, while PF shows four weight loss stages. Flynn‐Wall‐Ozawa calculation indicates that BRP has thermal stability between IIR and PF. Thermogravimetric analysis–Fourier transform infrared (TGA‐FTIR) and pyrolysis–gas chromatography/mass spectrometry (GC/MS) were used to investigate the volatile products under nitrogen atmosphere. As expected, BRP shows combined thermal decomposition behavior of both IIR and PF. The degradation mechanism of BRP was proposed, which is not significantly influenced by the incorporation of PF. The application stability of BRP is worth to be noticed since the post‐cure effect, that is, the free radicals remained from vulcanization would cause additional cross‐linking when stored at 80°C to 120°C.     

Rapid and sensitive determination of free thiols by capillary zone electrophoresis with near‐infrared laser‐induced fluorescence detection using a new BODIPY‐based probe as labeling reagent

A CZE with near‐infrared (NIR) LIF detection method has been developed for the analysis of six low molecular weight thiols including glutathione, homocysteine, cysteine, γ‐glutamylcysteine, cysteinylglycine, and N‐acetylcysteine. For this purpose, a new NIR fluorescent probe, 1,7‐dimethyl‐3,5‐distyryl‐8‐phenyl‐(4'‐iodoacetamido)difluoroboradiaza‐s‐indacene was utilized as the labeling reagent, whose excitation wavelength matches the commercially available NIR laser line of 635 nm. The optimum procedure included a derivatization step of the free thiols at 45°C for 25 min and CZE analysis conducted within 14 min in the running buffer containing 16 mmol/L pH 7.0 sodium citrate and 60% v/v ACN. The LODs (S/N = 3) ranged from 0.11 nmol/L for N‐acetylcysteine to 0.31 nmol/L for γ‐glutamylcysteine, which are better than or comparable to those reported with other derivatization‐based CE‐LIF methods. As the first trial of NIR CE‐LIF method for thiol determination, the practical application of the proposed method has been validated by detecting thiols in cucumber and tomato samples with recoveries of 96.5–104.3%.     

Novel monofunctional and bifunctional boronic acid‐functionalized squarylium dyes as precolumn and on‐column labels for protein analysis by capillary electrophoresis with laser‐induced fluorescence

This article presents a continuous capillary electrophoresis with laser‐induced fluorescence (CE‐LIF) following spectral studies of the noncovalent interactions between novel Squarylium Boronic Acid 4 (SQ‐BA4) & Squarylium Diboronic Acid 2 (SQ‐DBA2) squarylium dyes and human serum albumin (HSA). Two protocols were used wherein the on‐column‐labeling protocol was found to be more sensitive than the precolumn one by showing a better enhancement in the peak area of the HSA–dye complex besides lower limits of detection (LODs) for HSA. Also, stability studies were conducted with or without HSA using precolumn‐labeling mode over one week exhibiting the superiority of SQ‐BA4 to SQ‐DBA2. Then, a mixture containing three model proteins, HSA, β‐lactoglobulin B, and transferrin, was labeled on‐column with both dyes and completely resolved by CE‐LIF after optimization of several parameters. Both dyes provided lower LODs for HSA than those of β‐lactoglobulin B and transferrin with higher sensitivities. In addition, the SQ‐BA4 dye showed again greater sensitivities with all the three proteins than SQ‐DBA2.     

Boron‐chelating fluorescent probe (BOPB) in the red region combined with CE‐LIF for the detection of NO in mice liver

Precise measurement of nitric oxide (NO) is of great importance to understand the function of NO in liver and the mechanism of liver injury. 8‐(3’,4’‐Diamino phenyl)‐3,5‐(2‐hydroxyphenyl)‐dimethylene pyrrole (BOPB), a fluorescent probe in the red region (>600 nm) newly developed in our group, has good photostability and excitation/emission wavelength of 622/643 nm matching well with commercial 635 nm semiconductor laser of CE‐LIF detection. Therefore, BOPB was used in CE‐LIF for the determination of NO in mice liver. Both derivatization and separation conditions were optimized. Derivatization reaction of BOPB and NO was carried out in pH 7.4 PBS buffer at 35°C for 12 min and the separation of NO derivative of BOPB (BOPB‐T) was achieved within 7.0 min in pH 9.0 running buffer containing 15 mM H₃BO₃–NaOH and 15 mM SDS. Good linearity was found in the range of 1.0 × 10^?9–5.0 × 10^?7 M with the LOD of 0.02 nM. The proposed method was applied to the analysis of NO in real samples, and NO concentration was obviously increased in acute liver injury of mice. Compared to existing derivatization‐based CE‐LIF methods for NO, this method has lower LOD and less background interference owing to detection wavelength of BOPB in the red region.     

Synthesis, Characterization and Thermal Properties of Energetic Compounds Derived from 3-Amino-4-(tetrazol-5-yl)furazan

Five energetic compounds, 3,3‐bis(tetrazol‐5‐yl)‐4,4‐azofurazan (DTZAF), 3‐nitro‐4‐(tetrazol‐5‐yl)furazan (NTZF), hydrazinium 3‐amino‐4‐(tetrazol‐5‐yl)furazan (HATZF), triaminoguanidinium 3‐amino‐4‐(tetrazol‐ 5‐yl)furazan (TAGATZF) and guanylureaium 3‐amino‐4‐(tetrazol‐5‐yl)furazan (MATZF), were prepared using 3‐amino‐4‐(tetrazol‐5‐yl)furazan (ATZF) as starting material and their structures were characterized by FT‐IR, ¹H NMR, ¹³C NMR and elemental analysis. The properties of NTZF were estimated:density is 1.67 g/cm³, enthalpy of formation +415.41 kJ/mol and detonation velocity 8257.83 m/s. The main thermal properties of four compounds, DTZAF, HATZF, TAGATZF and MATZF, were analyzed by TG and DSC techniques and the results showed that their melting points are 251.9, 159.7, 205.4 and 211.4°C, respectively, and their first decomposition temperatures are 256.7, 258.6, 231.7 and 268.6°C, respectively. The fact that their decomposition temperatures were over 230°C showed that they exhibit better thermal stability.     

Determination of micro‐RNA in cardiomyoblast cells using CE with LIF detection

Micro‐RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE‐LIF) using fluorescence‐labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA‐499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA‐499 was found when hybridization was conducted at 40°C in 50 mM Tris‐acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA‐499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA‐499 was completed within 1 h using CE‐LIF. These results showed the potential of CE for fast, specific, and sensitive high‐throughput analysis of low‐abundance miRNAs in cell extracts, biofluids, and tissues.     

Total lipids of the intramuscular tissue of fallow deer

In this study non-isothermal non-oxidative and oxidative TG analysis was applied as a method for determining the thermal stability of the total lipids extracted from raw and maturing intramuscular tissue of fallow deer (Cervus Dama Dama L.). The total lipids were extracted from intramuscular tissue, according to the Folch method and stored for nine months at +4 and −18°C. The changes in the thermal stability of the total lipids during non-oxidative and oxidative TG analysis were correlated with the lipid composition, i.e. fatty acid composition. The Flynn-Wall method was used to determine the values of the activation energy of thermal degradation of the total lipids in a defined mass loss range.     

Polyisobutylene‐based polyurethanes. IX. synthesis,characterization, and properties of polyisobutylene‐based poly(urethane‐ureas)

Polyisobutylene (PIB)‐based polyurethanes (PUs) exhibit unparalleled hydrolytic‐oxidative‐biologic stability and are melt processible, however, their mechanical (strength) properties are modest mainly due to insufficient H bonds. We posited and demonstrate that the ultimate properties of PIB‐PUs are enhanced, while their melt processibility is maintained, by the judicious introduction of urea linkages, i.e., strong bifurcated H bonds, in the chain. The incorporation of bifurcated H bonds in PIB‐PUs was achieved by using the conventional butane diol chain extender (CE) in combination with controlled amounts of amino alcohol as co‐chain extender (co‐CE). Polyurethanes containing both urethane and urea linkages are polyurethane‐ureas (PUU). Specifically, PIB‐PUUs prepared with PIB‐diol/MDI together with 80/20 mole % butane diol/amino butanol exhibited ～30 MPa tensile strength, ～550% elongation, ～80 Shore A hardness, and ～137 °C flow temperature. Other amino alcohols, i.e., amino ethanol, ‐propanol, and ‐hexanol, were less effective co‐CEs. ¹H‐NMR and FT‐IR spectroscopies indicate the presence of bifurcated H bonds in PIB‐PUUs prepared with CE/co‐CE combinations. Characterization by differential scanning calorimetry, thermogravimetric analysis, dynamic mechanical thermal analysis, and creep experiments also suggest bifurcated H bonds in PIB‐PUU. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 2361–2369     

Near‐infrared electrochromic poly(aryl ether)s based on isolated electroactive tetraphenyl‐p‐phenylenediamine moieties

A series of near‐infrared (NIR) electrochromic aromatic poly(aryl ether)s containing N,N,N′,N′‐tetraphenyl‐p‐phenylenediamine (TPPA) moieties in the backbone were prepared from the high‐temperature polycondensation reactions of a biphenol monomer, 2,5‐bis(diphenylamino)hydroquinone, with difluoro compounds. The obtained polymers were readily soluble in many organic solvents and showed useful levels of thermal stability associated with high glass‐transition temperatures (182–205 °C) and high char yields (higher than 40% at 800 °C in nitrogen). The polymer films showed reversible electrochemical oxidation with high contrast ratio both in the visible range and NIR region, and also exhibited high coloration efficiency (CE), low switching time, and stability for electrochromic operation. The polyether TPPA‐a thin film revealed good CE in visible (CE = 217 cm²/C) and NIR (CE = 192 cm²/C) region with reversible electroactive stability (over 500 times within 5% loss relative to its initial injected charge). © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 5378–5385, 2009     

The stability of doxorubicin and ldarubicin in plasma and whole blood

Summary The stability of doxorubicin and idarubicin was studied in spiked human EDTA-, heparin-plasma and whole blood at room temperature (RT) and at +4°C. The analysis was performed by capillary electrophoresis (CE). Under each sel of conditions, doxorubicin and idarubicin concentrations decreased rapidly with time; the decrease was insignificantly more rapid with doxorubicin. Neither doxorubicinol nor idarubicinol were detectable. In order to obtain comparable plasma levels, doxorubicin and idarubicin containing blood samples should be centrifuged and stored at −20°C immediately.     

Entrapment of ethyl vanillin in calcium alginate and calcium alginate/poly(vinyl alcohol) beads

Electrostatic extrusion was applied to the encapsulation of 3-ethoxy-4-hydroxybenzaldehyde (ethyl vanillin) in calcium alginate and calcium alginate/poly(vinyl alcohol) beads. The calcium alginate/poly(vinyl alcohol) hydrogel spheres were formed after contact with the cross-linker solution of calcium chloride, followed by the freeze-thaw method for poly(vinyl alcohol) gel formation. The entrapment of aroma in beads was investigated by FTIR and thermal analysis (thermogravimetry/differential thermal gravimetry; TGA/DTG). The mass loss in the temperature range of 150?C300°C is related to degradation of the matrix and the release of ethyl vanillin. According to the DTG curve, the release of ethyl vanillin occurs at about 260°C. TGA measurements of the stored samples confirmed that formulations were stable for a period of one month. FTIR analysis provides no evidence for chemical interactions between flavour and alginate that would alter the nature of the functional groups in the flavour compound.     

Synthesis and characterization of a fluorinated polyanhydride

A fluorinated aromatic polyanhydride ( B ) was synthesized from the melt condensation of mixed anhydrides of 4,4′‐(hexafluoroisopropylidene)bis benzoic acid. Although the mixed anhydride from acetic anhydride yielded only a mixture of oligomers (weight‐average molecular weight < 2000), higher weight‐average molecular weight materials in the range of 15,000–18,000 were obtained with trifluoroacetic anhydride. Polymer B was soluble in chloroform and tetrahydrofuran, had a relatively high glass‐transition temperature of 176 °C with no melting point detected to 310 °C, and showed excellent thermal stability (5% weight loss observed at 380 °C by thermogravimetric analysis). The hydrolytic degradation of the fluorinated polyanhydride in a 0.1 M phosphate buffer of pH 7.4 at 37 °C was initially zero‐order, with 35% degradation occurring in 10 days. Loss of film integrity following that led to accelerated degradation, and almost complete dissolution was observed by the 16th day. The stability of the fluorinated polyanhydride in the solid state and in the solvent tetrahydrofuran was also evaluated. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 3027–3036, 2002     

Capillary electrophoresis‐laser‐induced fluorescence detection of rat brain catecholamines with microwave‐assisted derivatization

In this study, a rapid and sensitive method is described for the catecholamines detection in rat brain. CE with LIF detection for the determination of FITC derivatized catecholamines (dopamine, epinephrine, and norepinephrine) was demonstrated. Conventional water bath and microwave‐assisted derivatization methods were employed and a significant reduction in the derivatization time from 2 h for the conventional water bath at room temperature (ca. 25°C) to 2 min for the microwave‐assisted derivatization was achieved. Online sample concentration of field‐amplified sample stacking (FASS) method was employed to achieve higher sensitivities (the detection limits obtained in the normal injection mode ranged from 2.6 to 4.5 ng L^?1 and in the FASS mode ranged from 22 to 34 pg L^?1). Furthermore, this microwave‐assisted derivatization CE–LIF method successfully determined catecholamines in rat brain with as low as 100 ng L^?1 (FASS mode) to 10 μg L^?1 (normal injection mode). This CE–LIF method provided better detection ability when compared to the best reports on catecholamines analyses.     

The influence of silicone shell on double-layered microcapsules in intumescent flame-retardant natural rubber composites

The mechanisms of the thermal degradation of polyhedral oligomeric octaphenylsilsesquioxane (OPS), octa(nitrophenyl)silsesquioxane (ONPS), and octa(aminophenyl)silsesquioxane (OAPS) were investigated. The –NO₂ or –NH₂ substituents on the phenyl group affected the mechanism of the POSS thermal degradation. The thermal stabilities of OPS, ONPS, and OAPS were characterized by TG and FTIR. Thermal degradation of OPS included mainly the degradation of caged polyhedral oligomeric silsesquioxane structures and phenyl groups. Nitro or amino substituents decreased its thermal stability. The thermal degradation processes of OPS, ONPS, and OAPS differed. Phenyl groups and cyclobutadiene were observed in the OPS degradation products. Oxygen radicals that caused intensive CO₂ release between 350 and 450 °C were generated by the degradation of ONPS –NO₂. OAPS released mainly aminophenyl groups at 370 °C, whereas a small number of phenyl groups decomposed at 500 °C. The OAPS reactivity could enhance the thermal stability of POSS structure in the polyimide OAPS composites.     

Vinyl‐Type Polymerization of Norbornene Dicarboxylic Acid Dialkyl Esters

Summary: Poly(cis‐norbornene‐exo‐2,3‐dicarboxylic acid dialkyl esters) (alkyl = Me, Et, Pr, Bu, Pen, and Hex) are synthesized as a vinyl‐type with a palladium(II ) catalyst in high yield from easily prepared, pure exo‐monomers. The polymers show good solubility in common organic solvents and excellent thermal stability up to 330 °C. The polymers with alkyl groups larger than methyl exhibit a two‐step thermal degradation profile of an initial thermal degradation of side‐chains starting at 350 °C, followed by thermal degradation of the norbornene backbone starting at ca. 430 °C. The glass transition temperature decreases and the mechanical flexibility increases as the alkyl length of the side‐chain increases.

Normalized loss shear modulus (G″) versus temperature for polynorbornene dicarboxylic acid dialkyl esters.     

A high‐sensitivity LIF detector with silver mirror coating detection window and small‐angle optical deflection from collinear system for CE

An LIF detector was integrated into a CE system based on silver mirror coating detection window and small‐angle optical deflection from collinear configuration. For this detection scheme, the incident light beam was focused on capillary through the edge of a lens, resulting in a small deflection angle that deviated 18° from the collinear configuration. Meanwhile, the excitation light and emitted fluorescence were effectively reflected by silver mirror coating at the detection window. The fluorescence was collected through the center of the same lens and delivered to a PMT in the vertical direction. In contrast to conventional collinear LIF detection systems, the fluorescence intensity was greatly enhanced and the background level was significantly eliminated. FITC and FITC‐labeled amino acids were used as model analytes to evaluate the performance with respect to design factors of this system. The limit LOD was estimated to be 0.5 pM for FITC (S/N = 3), which is comparable to that of optimized confocal LIF systems. All the results indicate that the proposed detection scheme will be promising for development of sensitive and low‐cost CE system.     

Novel species of soluble thermally stable poly(keto ether ether amide)s: preparation,characterization, and properties

A new diamine containing one keto and four ether groups was prepared through a three‐step reaction: first, hydroquinone was reacted with 1‐fluoro‐4‐nitrobenzene and 4‐(4‐nitrophenoxy) phenol was obtained. The next step was reduction of nitro group to amino group in which 4‐(4‐aminophenoxy) phenol was prepared. In the final step, the new diamine named as bis(4‐(4‐(4‐aminophenoxy)phenoxy)phenyl) methanone was synthesized through reaction of the later compound with 4,4′‐difluoro benzophenone. All prepared materials were fully characterized by spectroscopic methods and elemental analysis. Novel species of poly(keto ether ether amide)s were synthesized via polymerization reaction of the diamine with different diacid chlorides including terephthaloyl chloride, isophthaloyl chloride, and adipoyl chloride. All polyamides were characterized, and their properties such as thermal behavior, thermal stability, solubility, viscosity, water uptake, and crystallinity were investigated and compared together. The glass transition temperatures of the polymers were about 204–232°C, and their 10% weight losses were in the range of 396–448°C. Polymers showed high thermal stability and enhanced solubility that mainly resulted from incorporation of the diamine structure containing keto, ether, and aromatic units into polyamide backbones. Copyright © 2014 John Wiley & Sons, Ltd.