Evaluation of single-base substitution rate in DNA by affinity capillary electrophoresis

Capillary electrophoretic separation of 60 mer single-stranded DNA (ssDNA) and a single-base-substituted ssDNA was demonstrated using a size- and composition-controlled poly(ethylene glycol)-oligodeoxyribonucleotide block copolymer (PEG-b-ODN) as an affinity ligand. Under appropriate conditions, PEG-b-ODN and ssDNA with a complementary sequence formed a reversible complex via hybridization during the electrophoresis, while the copolymer did not interact with the single-base-substituted ssDNA. The copolymer's PEG length determined the electrophoretic mobility of the ssDNA; upon formation of the complex, the electrically neutral PEG added hydrodynamic friction to ssDNA. Simultaneously using two types of PEG-b-ODN copolymers whose PEG segments were of different lengths, we achieved the complete separation of the 60 mer ssDNA, its single-base-substituted ssDNA, and impurities. This method was sensitive enough to quantify a slight amount (approximately 1%) of the single-base-substituted ssDNA. The present results suggest that our approach is applicable to quantitative detection of minor genotypes.     

Affinity capillary electrophoretic DNA separation using PEG-oligodeoxyribonucleotide block copolymers: relationship between peak resolution and affinity strength

Capillary electrophoretic separation of chemically synthesized ssDNA and a single-base-substituted one (normal and mutant ssDNA, respectively) was demonstrated using a PEG-oligodeoxyribonucleotide block copolymer (PEG-b-ODN) as an affinity ligand. When the base sequence of PEG-b-ODN was designed to be complementary to part of normal ssDNA including the base-substituted site, the electrophoretic mobility of normal ssDNA significantly decreased whereas that of mutant ssDNA slightly changed. Resolution of the separation strongly depended on the ODN length of the copolymer, the capillary temperature, and the Mg2+ concentration in the running buffer, indicating that the retardation of migration of normal ssDNA was induced by the reversible hybridization with PEG-b-ODN. It was found that the dissociation constant (K(d)) of the duplex between normal ssDNA and the affinity probe ODN should be smaller than 10(-6) M to achieve the good peak separation. In addition, we calculated the mobility of the complex (mu(C)) between normal ssDNA and PEG-b-ODN using a two-state model. The base sequence of affinity probe ODN appropriate to achieve the sufficient resolution will be predicted on the basis of the mu(C) and K(d )values.     

Analysis of neutral surfactants by non-aqueous capillary electrophoresis using an electroosmotic flow reversal

The separation of KM 20, that is in fact a mixture of non-ionic surfactants, was carried out by non-aqueous capillary electrophoresis. This complex mixture resulting from the condensation of ethylene oxide with fatty alcohols does not have chromophoric moieties. So, we analysed it after derivatization by means of 3,5-dinitrobenzoyl chloride. The proposed approach is based both on the formation of complexes with alkaline or ammonium cations in methanol and on the utilisation of a positively charged capillary. From a comparative study on the capillary treatment procedure, we used hexadimethrine bromide as electroosmotic flow reverser in order to obtain both repeatable analyses and good resolutions of the largest KM 20 oligomers. Then, among the five cations used to form complexes with KM 20, we pointed out that ammonium cation led to the best resolutions. Moreover, we evidenced that the counter-ion of this cation had a great influence on resolution because it modified the magnitude of electroosmotic flow. Ion pair formation that is more or less strong between ammonium and its counter-ion was involved in this variation of electroosmotic flow. So, we calculated the association constants for various ammonium salts in methanol. Then, using ammonium chloride as background electrolyte, we optimised the concentration of this salt, in methanol, in order to reach the optimal separation of KM 20 oligomers. Thus, a baseline separation was obtained by using 6 x 10(-2) mol/L NH4Cl as running electrolyte. In these conditions, we separated, in about 30 min, more than 30 oligomers of KM 20. The distribution of these oligomers that was determined from the optimal separation, appeared consistent with that obtained from HPLC analyses. Indeed, we determined that the mean ethoxylation number was equal to 18 while its real value is equal to 20.     

反向电渗流非水毛细管电泳法快速测定微量赤霉素

赤霉素是一类重要的植物激素,是结构相似的弱酸性二萜类化合物。针对毛细管电泳法分离、测定赤霉素速度慢、效率不佳等问题,该文发展了一种可快速测定微量活性赤霉素的非水毛细管电泳-紫外检测法。以聚环氧乙烷动态涂布毛细管,利用正离子使电渗流反向,将含10 mmol/L醋酸铵的甲醇-水(95∶5,v/v)作为缓冲体系,在0.08%(v/v)醋酸含量下,分离8种内源性赤霉素。结果表明,赤霉素出峰时间的相对标准偏差(RSD)≤2.1%(日内)或≤4.3%(日间),峰面积的RSD≤4.5%(日内)或≤6.9%(日间),检出限(S/N=3)为1.04~2.20 mg/L,相关系数为0.998 2~0.999 3,回收率为87.2%~93.5%。该方法简单、快速、稳定,以含醋酸铵的甲醇水溶液为缓冲体系,预先考虑了质谱测定的需要,可用于实际样品如麦芽中赤霉素的分析,具有一定的应用价值。     

Precise synthesis of olefin block copolymers using a syndiospecific living polymerization system

This feature article summarizes the synthesis of novel olefin block copolymers using fast syndiospecific living homo- and copolymerization of propylene, higher 1-alkene, and norbornene with ansa-fluorenylamidodimethyltitaniumbased catalyst according to the authors’ recent results. The catalytic synthesis of monodisperse polyolefin and olefin block copolymer was also described using this living system.     

Photocrosslinkable polynorbornene-based block copolymers with enhanced dielectric and thermal properties

Block copolymers poly(endo-N-3,5-bis(trifluoromethyl)biphenyl-norbornene-pyrrolidine)-block-poly(exo-N-(cinnamoyloxyethyl)-7-oxanorborn-5-ene-2,3-dicarboximide) (endo-PTNP-b-exo-PCONBI) and poly(exo-N-3,5-bis(trifluoromethyl)biphenyl-norbornene-pyrrolidine)-block-poly(exo-N-(cinnamoyloxyethyl)-7-oxanorborn-5-ene-2,3-dicarboximide) (exo-PTNP-b-exo-PCONBI) were synthesized by ring-opening metathesis polymerization. The endo- or exo-PTNP served as the high dielectric functional chain, and exo-PCONBI acted as the crosslinking segment. The endo-PTNP-b-exo-PCONBI, in which endo-PTNP has a high content of trans double bond and adopts isotactic configuration, shows a dielectric constant (ε) of 15.5, whereas exo-PTNP-b-exo-PCONBI, in which exo-PTNP has 67% trans double bonds and atactic microstructure, displays relatively low ε of 7.1. The cinnamate groups in exo-PCONBI were crosslinked to form three-dimensional network by cycloaddition reaction under UV irradiation. Exposed to UV-light for 10 min, the cinnamate group in polymer films has a crosslinking conversion of 36%, as determined by UV-Vis absorption measurements. By photocrosslinking, the polymer film has an increased ε of 16.6, a dielectric loss of 0.03, an elevated glass-transition temperature of 137 °C, and an enhanced decomposition temperature of 405 °C, compared to those of polymer films without irradiation.     

Detection of single-base mutation by affinity capillary electrophoresis using a DNA-polyacrylamide conjugate

We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis.     

Rapid analysis of alkylphosphonate drugs by capillary zone electrophoresis using indirect ultraviolet detection

A rapid capillary electrophoretic method for the analysis of three alkylphosphonate drugs (i.e. fosfomycin disodium (FOS), clodronate disodium (CLO) and alendronate sodium (ALN)) was developed by using multiple probe BGE and indirect UV detection. BGE containing 30 mM benzoic acid, 5 mM salicylic acid and 0.5 mM CTAB (pH 3.8), temperature of 30°C, applied voltage of ?30 kV and detection at 220 nm provided baseline separation of all analytes (resolution (R)>2.2) in 3.2 min. EOF reversal by addition of CTAB and negative voltage polarity leading to the co‐EOF flow and short analysis time. Two probe BGE greatly improved peak symmetry. The method showed good linearity (r²>0.999 in ranges of 20–1000 μg/mL for FOS, 100–1000 μg/mL for CLO and 100–750 μg/mL for ALN) repeatablitiy (RSD<2.15%), recovery (99.3–101.1%) and sensitivity (LOD<50 μg/mL). Freshly prepared BGE and sample solutions are essential for the method precision and accuracy. This new method can be utilized for routine analysis of FOS, CLO and ALN in dosage forms because of its efficiency, reliability, speed and simplicity.     

Mobility-based selective on-line preconcentration of proteins in capillary electrophoresis by controlling electroosmotic flow

A simple method to perform selective on-line preconcentration of protein samples in capillary electrophoresis (CE) is described. The selectivity, based on protein electrophoretic mobility, was achieved by controlling electroosmotic flow (EOF). A short section of dialysis hollow fiber, serving as a porous joint, was connected between two lengths of fused silica capillary. High voltage was applied separately to each capillary, and the EOF in the system was controlled independently of the local electric field intensity by controlling the total voltage drop. An equation relating the EOF with the total voltage drop was derived and evaluated experimentally. On-line preconcentration of both positively charged and negatively charged model proteins was demonstrated without using discontinuous background electrolytes, and protein analytes were concentrated by approximately 60-200-fold under various conditions. For positively charged proteins, positive voltages of the same magnitude were applied at the free ends of the connected capillaries while the porous joint was grounded. This provided a zero EOF in the system and a non-zero local electric field in each capillary to drive the positively charged analytes to the porous joint. CE separation was then initiated by switching the polarity of the high voltage over the second capillary. For negatively charged proteins, the procedure was the same except negative voltages were applied at the free ends of the capillaries. Mobility-based selective on-line preconcentration was also demonstrated with two negatively charged proteins, i.e. beta-lactoglobulin B and myoglobin. In this case, negative voltages of different values were applied at the free ends of the capillaries with different values, which provided a non-zero EOF in the system. The direction of EOF was the same as that of the electrophoretic migration velocities of the protein analytes in the first capillary and opposite in the second capillary. By controlling the EOF, beta-lactoglobulin B, which has a higher mobility, could be concentrated over 150-fold with a 15 min injection while myoglobin, which has a lower mobility, was eliminated from the system.     

Moderation of the electroosmotic flow in capillary electrophoresis by chemical modification of the capillary surface with tentacle-like oligourethanes

The surface chemistry of the inner wall of fused-silica capillaries is one important means to control the magnitude as well as the direction of the electroosmotic flow and the adsorption activity. A method was developed to change the surface characteristics of fused-silica capillaries by binding tentacle-like oligourethane groups onto the inner surface. The electroosmotic flow at a buffer pH of 6-9 was reduced by 15 to 40% compared to that in a bare fused-silica tubing, dependent on the type of coating. Sample adsorption is diminished at the same time resulting in a separation of proteins with higher resolution and good migration time precision. At a pH below 4.5 the electroosmotic flow is reversed into the anodic direction, which offers further possibilities for the separation of positively charged analytes as demonstrated for the separation of aromatic and biogenic amines.     

Aggregation and solubilization of organic solvents and petrol/gasoline in water mediated by block copolymers

A series of AB and ABA block copolymers of pDEGMEMA-b-pCHMA and pCHMA-b-pDEGMEMA-b-pCHMA cyclohexyl methacrylate (CHMA) and di(ethylene glycol) methyl ether methacrylate (DEGMEMA) with M_n ranging between 18,000 and 50,000 g mol⁻¹ and PDI = 1.09-1.32 were prepared via copper(I) mediated living radical polymerization with pyridylmethanimine ligands. Aggregation properties were investigated using a combination of ¹H NMR, dynamic and static light scattering. For comparative purposes poly(CHMA) and poly(DEGMEMA) homopolymers were prepared. The CAC values estimated for the di- and triblock copolymers soluble in cyclohexane are lower than 0.005 g L⁻¹ whereas the values found for block copolymers in methanol solutions are less than 0.070 g L⁻¹. DLS analysis showed the presence of micellar aggregates with diameters ranging from 25 to 40 nm with particle polydispersity indexes between 0.003 and 0.183. The pCHMA-b-pDEGMEMA-b-pCHMA micelles solubilized the aqueous phase in petrol/gasoline. The block copolymer-based micelles incorporate water within their hydrophilic domains, potentially overcoming a number of practical problems such as the formation of biphasic mixtures in solvent blends due to undesired water accumulation.     

Determination of molecular parameters of NR/PU block copolymers by transport studies

The equilibrium swelling experiments of block copolymers based on natural rubber (NR) as soft segment and polyurethane (PU) derived from toluene diisocyanate (TDI) and 1,3-butane diol (1,3-BDO) as hard segment were performed in various solvents. Block copolymers with different NCO/OH ratio, viz., from 1.26 to 1.96 were employed in this study. The crosslink densities are found to vary with the NCO/OH ratio employed in the synthesis of the samples. The sorption behaviour is also found to vary with the NCO/OH ratio. The equilibrium swelling experiments have been used to determine the molecular parameters such as molecular mass between crosslinks, crosslink density, polymer-solvent interaction parameter, etc. The equilibrium swelling values were also used to calculate the solubility parameter values of the block copolymers.     

Determination of electroosmotic flow mobility with a pressure-mediated dual-ion technique for capillary electrophoresis with conductivity detection using organic solvents

A method is described for the indirect determination of the mobility of the electroosmotic flow (EOF), which can be carried out within a few minutes even for very low mobilities. It is independent of the direction of the EOF. It is based on the comparison of the measured mobilities of two oppositely charged reference ions (tetraphenylphosphonium and tetraphenylborate) with given mobilities in different organic solvents (methanol, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, propylene carbonate) at ionic strengths between 5 and 50 mM. The method is based on the sequential movement of the reference ions in a three-step process: first by a laminar flow to a certain position in the separation capillary, followed by electromigration due to application of voltage, and pressurised migration towards the detector. In this way the total mobilities of the reference ions can be determined from their residence times, and the difference to their known actual mobilities gives the mobility of the EOF. The method avoids misinterpretations caused by system- and eigen-peaks, which often bias the results especially when a conductivity detector is used. The method is suitable for all solvents, and is an advantage especially for organic and mixed aqueous–organic background electrolytes with high UV absorbance.     

Interaction of albumins and heparinoids investigated by affinity capillary electrophoresis and free flow electrophoresis

A fast and precise affinity capillary electrophoresis (ACE) method has been applied to investigate the interactions between two serum albumins (HSA and BSA) and heparinoids. Furthermore, different free flow electrophoresis methods were developed to separate the species which appears owing to interaction of albumins with pentosan polysulfate sodium (PPS) under different experimental conditions. For ACE experiments, the normalized mobility ratios (∆R/R_f), which provided information about the binding strength and the overall charge of the protein‐ligand complex, were used to evaluate the binding affinities. ACE experiments were performed at two different temperatures (23 and 37°C). Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, the interactions can already be observed at low mg/L concentrations (3 mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The additional signals also appeared at higher concentrations at 37°C. Nevertheless, in most cases the binding data were similar at both temperatures. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The free flow electrophoresis methods have confirmed and completed the ACE experiments.     

Amphiphilic block copolymers directed synthesis of mesoporous nickel-based oxides with bimodal mesopores and nanocrystal-assembled walls

Mesoporous late-transition metal oxides have great potential in applications of energy,catalysis and chemical sensing due to their unique physical and chemical properties.However,their synthesis via the flexible and scalable soft-template method remain a great challenge,due to the weak organic-inorganic interaction between the frequently used surfactants(e.g.,Pluronic-type block copolymers) and metal oxide precursors,and the low crystallization temperature of metal oxides.In this study,ordered mesoporous NiO with dual mesopores,high surface area and well-interconnected crystalline porous frameworks have been successfully synthesized via the facile solvent evaporation-induced co-assembly(EICA) method,by using lab-made amphiphilic diblock copolymer polystyrene-b-poly(4-vinylpyridine)(PS-b-P4 VP) as both the structure-directing agent(the soft template) and macromolecular chelating agents for nickel species,THF as the solvent,and nickel acetylacetonate(Ni(acac)2) as inorganic precursor.Similarly,by using Ni(acac)2 and Fe(acac)3 as the binary precursors,ordered mesoporous Fedoped NiO materials can be obtained,which have bimodal mesopores of large mesopores(32.5 nm) and secondary mesopores(4.0-11.5 nm) in the nanocrystal-assembled walls,high specific surface areas(～74.8 m~2/g) and large pore value(～0.167 cm~3/g).The obtained mesoporous Fe-doped NiO based gas sensor showed superior ethanol sensing performances with good sensitivity,high selectivity and fast response-recovery dynamics.     

Microphase separation at the surface of block copolymers, as studied with atomic force microscopy

Atomic force microscopy (AFM) is used to study the phase separation process occurring in block copolymers in the solid state. The simultaneous measurement of the amplitude and the phase of the oscillating cantilever in the tapping mode operation provides the surface topography along with the cartography of the microdomains of different mechanical properties. This technique thus allows to characterize the size and shape of those microdomains and their organization at the surface (e.g. cubic lattice spheres, hexagonal lattice of cylinders, or lamellae). In this study, a series of symmetric triblock copolymers made of a inner elastomeric sequence (poly(butadiene) or poly(alkylacrylate)) and two outer thermoplastic sequences (poly(methylmethacrylate)) is analyzed by AFM in the tapping mode. The microphase separation and their morphology are essential factors for the potential of these materials as a new class of thermoplastic elastomers. Special attention is paid to the control of the surface morphology, as observed by AFM, by the molecular structure of the copolymers (volume ratio of the sequences, molecular weight, length of the alkyl side group) and the experimental conditions used for the sample preparation. The molecular structure of the chains is completely controlled by the synthesis, which relies on the sequential living anionic polymerization of the comonomers. The copolymers are analyzed as solvent-cast films, whose characteristics depend on the solvent used and the annealing conditions. The surface arrangement of the phase-separated elastomeric and thermoplastic microdomains observed on the AFM phase images is discussed on the basis of quantitative information provided by the statistical analysis by Fourier transform and grain size distribution calculations.     

Ultraslow dynamics in asymmetric block copolymers with nanospherical domains

Low shear rate and low frequency measurements focused on the extremely slow dynamics of a three-dimensional body-centered cubic (BCC) structure of an asymmetric block copolymer under nanophase-separated conditions. The material studied was poly(styrene-b-ethylene-co-butylene-b-styrene) swollen in a hydrocarbon oil selective for the midblock. Transient viscosities during start-up of shear flow at extremely low shear rates are governed by very long relaxation times and by a modulus that is nearly the same as the plateau modulus obtained from oscillatory shear experiments. Only at extremely low shear rates a zero shear viscosity could be attained. Its very high value is close to the viscosity calculated from stress relaxation experiments. The steady shear viscosity decreases by several orders of magnitude when increasing the shear rate. SAXS experiments on samples sheared even at very low rates indicated loss of the BCC order that was present in the annealed samples before shearing. The SAXS profile recorded on such a sample showed a first-order maximum followed by a broad shoulder indicating a liquid-like short-range order of PS nanospheres in the swollen EB matrix.