Mixed triblock copolymers used as DNA separation medium in capillary electrophoresis

A polymer solution, formed by mixing two polyoxybutylene-polyoxyethylene-polyoxybutylene (BEB) triblock copolymers (B10E270B10 and B6E46B6), was tested as a new separation medium for double-stranded DNA separation in capillary electrophoresis. The mixture of B10E270B10 and B6E46B6 has a viscosity-adjustable property and a dynamic coating ability, which makes the medium very easy to handle. The performance of the mixture on the DNA separation is greatly affected by the mass ratio of the two constituents. There is a minimum amount of concentration for B10E270B10, below which the medium will lose its performance. The addition of B6E46B6 increases both the selectivity and the separation efficiency. The optimal concentration, with 3% (w/v) B10E270B10 and 5% (w/v) B6E46B6, is determined with the consideration of both speed and resolution. A resolution of 1.3 was achieved on the separation of 123/124 base pairs in the pBR322/HaeIII digest within 20 min by using a 10 cm column of 75 microm I.D., demonstrating the potential use of mixtures of amphiphilic block copolymers as an effective DNA separation medium.     

Separation of double-stranded DNA fragments in plastic capillary electrophoresis chips by using E99P69E99 as separation medium.

The separation of double-stranded DNA (dsDNA) fragments in polymethylmethacrylate (PMMA) capillary electrophoresis (CE) chips by using E99P69E99 as a separation medium has been demonstrated. The PMMA CE chips were simply manufactured by micromachining and adhesive tape sealing. To make the separation channel compatible with the separation medium, a dynamic nonionic surfactant coating procedure was developed, which made the plastic separation channel sufficiently hydrophilic to allow the separation medium to fill the channel by capillary action. Subsequent separation of DNA fragments was successful with a separation efficiency of the order of 10(4) theoretical plates over an effective separation distance of 1.5 cm. By using an applied electric field strength of 200 V/cm, the separation of low DNA mass ladder was completed within 5 min. The simple coating procedure, together with the self-assembled viscosity-adjustable separation medium, should be useful to meet some of the essential requirements for developing single-use disposable CE chips. Coating the channels with polymer blends of PMMA and the separation medium also showed promise.     

Novel hydroxyethylcellulose-graft-poly acrylamide copolymer for separation of double-stranded DNA fragments by CE

A novel separation medium, hydroxyethylcellulose-graft-polyacrylamide (HEC-g-PAM) synthesized by atom transfer radical polymerization (ATRP), used for dsDNA separation by CE is presented. The separation performance of HEC-g-PAM, which has the same graft density and different graft length, has been investigated in Tris-boric acid-EDTA (TBE) buffer solvent mixtures. The temperature-dependent rheological behavior of HEC-g-PAM was also studied by steady-shear rheometry. The results showed that dsDNA fragments between 72 and 1353 bp was achieved with a 30 cm effective capillary length at 150 V/cm using this type of graft copolymer as a separation medium in bare fused-silica capillaries, and separation improvement is obtained in HEC-g-PAM compared with HEC and poly(dimethylacrylamide (PDMA).     

Hydroxyethylcellulose-graft-poly (N,N-dimethylacrylamide) copolymer as a multifunctional separation medium for CE

A new multifunctional separation medium, hydroxyethylcellulose-graft-poly (N,N-dimethylacrylamide) copolymer synthesized in our laboratory for application in both basic protein separation and dsDNA separation by CE, is presented in this paper. As a noncovalent coating, this medium showed a powerful capability in resisting basic protein adsorption. Highly efficient and rapid protein separation had been obtained at four different pH values. Meanwhile, the 11 fragments of the dsDNA sample could be baseline separated using this grafted copolymer as sieving matrix at an appropriate concentration.     

Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking

Phospholipid additives are a cost-effective medium to separate deoxyribonucleic acid (DNA) fragments and possess a thermally-responsive viscosity. This provides a mechanism to easily create and replace a highly viscous nanogel in a narrow bore capillary with only a 10 °C change in temperature. Preparations composed of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble, forming structures such as nanodisks and wormlike micelles. Factors that influence the morphology of a particular DMPC–DHPC preparation include the concentration of lipid in solution, the temperature, and the ratio of DMPC and DHPC. It has previously been established that an aqueous solution containing 10% phospholipid with a ratio of [DMPC]/[DHPC] = 2.5 separates DNA fragments with nearly single base resolution for DNA fragments up to 500 base pairs in length, but beyond this size the resolution decreases dramatically. A new DMPC–DHPC medium is developed to effectively separate and size DNA fragments up to 1500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1500 base pairs. This increase in the upper size limit is accomplished using commercially available phospholipids at an even lower material cost than is achieved with the 10% preparation. The separation additive is used to evaluate size markers ranging between 200 and 1500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these self-assembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix of low viscosity. DNA sample stacking is facilitated with longer injection times without sacrificing separation efficiency.     

A block copolymer covalent coating acting as surfactants in separation of 2-[hydroxy(4-nitrophenyl)methyl]-cyclopent-2-enone and 4-nitrobenzaldehyde by capillary electrophoresis

An innovative block copolymer capillary coating P(MAn-alt-St)₁₂₇-b-PSt₅₉₂, synthesized by maleic anhydride and styrene, was developed as a new kind of coating for capillary electrophoresis. The covalent bond coating was effectively applied in the separation of raw material (4-nitrobenzaldehyde) and production (2-[hydroxy(4-nitrophenyl)methyl]-cyclopent-2-enone) in a Baylis-Hillman reaction using ammonium acetate with 20% tetrahydrofuran (v/v) as the buffer solution. Electroosmotic flow measurement gave an instantly stable value after 70 times continued injection in 5 days and showed that P(MAn-alt-St)₁₂₇-b-PSt₅₉₂ coatings could suppress electroosmotic flow effectively compared with the bare capillary. The effects of tetrahydrofuran and the pH of buffer on the separation were investigated. The characteristics of the coatings to form micelles similar to surfactants were detected by atomic force microscopy. Moreover, the superiority of this coating was further applied in the separation of four aromatic amines.     

A new quasi-interpenetrating network formed by poly(N-acryloyl-tris-(hydroxymethyl)aminomethane and polyvinylpyrrolidone: separation matrix for double-stranded DNA and single-stranded DNA fragments by capillary electrophoresis with UV detection

The preparation of a new separation matrix, quasi-interpenetrating networks (quasi-IPNs) formed by poly(N-acryloyl-Tris) (poly(tris-A)) and PVP, and its application for dsDNA and ssDNA fragments separation by CE with UV detection, are presented. This new quasi-IPN exhibited high sieving performance, good dynamic coating ability, and low viscosity. Single-base resolutions of dsDNA fragments (Rs = 0.92 for 123/124 bp) and ssDNA fragments (Rs = 0.65 for 123/124 base, Rs = 0.48 for 309/310 base) were achieved by using the quasi-IPN of poly(tris-A)/PVP (2% + 2%) solution in a 31 cm effective length linear polyacrylamide (LPA)-coated column. Single-base separation of dsDNA fragments (Rs = 0.92 for 123/124 bp) was also obtained within 28 min in a 46.7 cm effective length bare column at higher 160 V/cm electric field strength by using the same quasi-IPN solution. The RSD of the migration time measured for each DNA fragments was less than 1.5% in the bare column for nine continuous runs. The effects of temperature and electric field strength on the DNA separation were also investigated.     

Block polyelectrolytes and colloidal stability

The interactions of sodium dodecyl sulfate (SDS) with poly(ethylene oxide)/poly(alkylene oxide) (E/A) block copolymers are explored in this study. With respect to the specific compositional characteristics of the copolymer, introduction of SDS can induce fundamentally different effects to the self-assembly behavior of E/A copolymer solutions. In the case of the E(18)B(10)-SDS system (E = poly(ethylene oxide) and B = poly(butylene oxide)) development of large surfactant-polymer aggregates was observed. In the case of B(20)E(610)-SDS, B(12)E(227)B(12)-SDS, E(40)B(10)E(40)-SDS, E(19)P(43)E(19)-SDS (P = poly(propylene oxide)), the formation of smaller particles compared to pure polymeric micelles points to micellar suppression induced by the ionic surfactant. This effect can be ascribed to a physical binding between the hydrophobic block of unassociated macromolecules and the non-polar tail of the surfactant. Analysis of critical micelle concentrations (cmc(*)) of polymer-surfactant aqueous solutions within the framework of regular solution theory for binary surfactants revealed negative deviations from ideal behavior for E(40)B(10)E(40)-SDS and E(19)P(43)E(19)-SDS, but positive deviations for E(18)B(10)-SDS. Ultrasonic studies performed for the E(19)P(43)E(19)-SDS system enabled the identification of three distinct regions, corresponding to three main steps of the complexation; SDS absorption to the hydrophobic backbone of polymer, development of polymer-surfactant complexes and gradual breakdown of the mixed aggregates.     

Depletion-induced surface alignment of asymmetric diblock copolymer in selective solvents

Phase separation of asymmetric diblock copolymer near surfaces in selective solvents is theoretically investigated by using the real-space version of self-consistent field theory (SCFT). Several morphologies are predicted and the phase diagram is constructed by varying the distance between two parallel hard surfaces (or the film thickness) W and the block copolymer concentration f(P). Morphologies of the diblock copolymer in dilute solution are found to change significantly with different film thicknesses. In confined systems, stable morphologies found in the bulk solution become unstable due to the loss of polymer conformation entropy. The vesicle phase region contracts when the repulsive interaction between the blocks is strong (strong segregation regime). The mixture of vesicles, rodlike and spherelike micelles and the mixture of vesicles and sphere-like micelles disappear in contrast to the weakly segregating regime. The walls strongly affect the phase separation of block copolymer in selective solvent, and the depletion layer near the surface contributes much to the micelle formation of the block copolymer. Interestingly, the self-assembled morphologies stay near the walls with the distance on the order of the radius of gyration of the block copolymer. The oscillation of the polymer distribution near the walls allows the surface phase separation to be observed due to the strong repulsion between the blocks A and B.     

Electrophoretic chip for high-fidelity fractionation of double-stranded DNA

We report the high fidelity, on-chip fractionation of selected segments from an electrophoretic flow of separated fragments. dsDNA fragments (10-330 base pairs (bp)) were initially separated using a 6.5 cm long channel with an electric field strength of 150 V/cm. As an example of the fractionation process, a target fragment of 20 bp was selected and extracted from the separation channel. The extraction was confirmed and evaluated by fluorescence imaging. High resolution and extraction fidelity were achieved by introducing new procedures for (i) extraction channel-blocking and (ii) segment transfer with cleaning. These procedures are necessary for the development of a practical, fully automated multitarget fractionation electrophoretic chip. A kind of CCD image processing method was introduced to monitor, control, and evaluate the procedure of fractionation. The resolution limits of the separation and extraction are discussed.     

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.     

Concentration gradient used in double-stranded DNA separation by capillary electrophoresis

Effects of concentration gradient on double-stranded DNA (dsDNA) separation by capillary electrophoresis are presented. By using a concentration gradient in the range between 0.8% and 3.2% for poly(N,N-dimethylacrylamide) (PDMA), the presence of a mesh-size gradient in the capillary could enhance the separation of larger size DNA fragments, better than that based on a single uniform concentration over the same capillary length. A decrease in the column length could make the gradient effect more obvious. An optimal capillary length could be achieved by using a judicious combination of the concentration gradient and the concentration range, yielding a maximum resolution for the system. The standard deviation of the migration time measured for each DNA fragment was less than 5% in ten continuous runs, suggesting that the gradient formed inside the column was quite stable.     

K+‐Responsive Block Copolymer Micelles for Targeted Intracellular Drug Delivery

In this work, a novel type of block copolymer micelles with K⁺‐responsive characteristics for targeted intracellular drug delivery is developed. The proposed smart micelles are prepared by self‐assembly of poly(ethylene glycol)‐b‐poly(N‐isopropylacry‐lamide‐co‐benzo‐18‐crown‐6‐acrylamide) (PEG‐b‐P(NIPAM‐co‐B18C6Am)) block copolymers. Prednisolone acetate (PA) is successfully loaded into the micelles as the model drug, with loading content of 4.7 wt%. The PA‐loaded micelles display a significantly boosted drug release in simulated intracellular fluid with a high K⁺ concentration of 150 × 10⁻³m , as compared with that in simulated extracellular fluid. Moreover, the in vitro cell experiments indicate that the fluorescent molecules encapsulated in the micelles can be delivered and specifically released inside the HSC‐T6 and HepG2 cells responding to the increase of K⁺ concentration in intracellular compartments, which confirms the successful endocytosis and efficient K⁺‐induced intracellular release. Such K⁺‐responsive block copolymer micelles are highly potential as new‐generation of smart nanocarriers for targeted intracellular delivery of drugs.     

Ultrafast, efficient separations of large-sized dsDNA in a blended polymer matrix by microfluidic chip electrophoresis: a design of experiments approach

Double-stranded (ds) DNA fragments over a wide size range were successfully separated in blended polymer matrices by microfluidic chip electrophoresis. Novel blended polymer matrices composed of two types of polymers with three different molar masses were developed to provide improved separations of large dsDNA without negatively impacting the separation of small dsDNA. Hydroxyethyl celluloses with average molar masses of ～27 kDa and ～1 MDa were blended with a second class of polymer, high-molar mass (～7 MDa) linear polyacrylamide. Fast and highly efficient separations of commercially available DNA ladders were achieved on a borosilicate glass microchip. A distinct separation of a 1-kb DNA extension ladder (200-40,000 bp) was completed in 2 min. An orthogonal design of experiments was used to optimize experimental parameters for DNA separations over a wide size range. We find that the two dominant factors are the applied electric field strength and the inclusion of a high concentration of low-molar mass polymer in the matrix solution. These two factors exerted different effects on the separations of small dsDNA fragments below 1 kbp, medium dsDNA fragments between 1 and 10 kbp, and large dsDNA fragments above 10 kbp.     

Aggregate morphologies of amphiphilic ABC triblock copolymer in dilute solution using self-consistent field theory

The complex microstructures of amphiphilic ABC linear triblock copolymers in which one of the end blocks is relatively short and hydrophilic, and the other two blocks B and C are hydrophobic in a dilute solution, have been investigated by the real-space implementation of self-consistent field theory (SCFT) in two dimensions (2D). In contrast to diblock copolymers in solution, the aggregation of triblock copolymers are more complicated due to the presence of the second hydrophobic blocks and, hence, big ranges of parameter space controlling the morphology. By tailoring the hydrophobic degree and its difference between the blocks B and C, the various shapes of vesicles, circlelike and linelike micelles possibly corresponding to spherelike, and rodlike micelles in 3D, and especially, peanutlike micelles not found in diblock copolymers are observed. The transition from vesicles to circlelike micelles occurs with increasing the hydrophobicity of the blocks B and C, while the transition from circlelike micelles to linelike micelles or from the mixture of micelles and vesicles to the long linelike micelles takes place when the repulsive interaction of the end hydrophobic block C is stronger than that of the middle hydrophobic block B. Furthermore, it is favorable for dispersion of the block copolymer in the solvent into aggregates when the repulsion of the solvent to the end hydrophobic block is larger than that of the solvent to the middle hydrophobic block. Especially when the bulk block copolymers are in a weak segregation regime, the competition between the microphase separation and macrophase separation exists and the large compound micelle-like aggregates are found due to the macrophase separation with increasing the hydrophobic degree of blocks B and C, which is absent in diblock copolymer solution. The simulation results successfully reproduce the existing experimental ones.     

毛细管电泳-限制性片段长度多态性分析检测胃癌H-ras基因点突变

建立一种毛细管电泳快速高效检测限制性内切酶酶切产物的方法, 使其更好地用于基因诊断. 以甲基纤维素(Methyl cellulose, MC)为筛分介质, 用pUC19 DNA/Msp I (Hpa II) Marker标准DNA片段为实验对象, 通过考察筛分介质的浓度、pH值、毛细管的温度和运行电压优化出分离小于600 bp的双链DNA片段的最适条件, 并将此方法应用于临床59例胃癌患者肿瘤组织H-ras基因12位密码子点突变情况的检测. MC是一种良好的筛分介质, 运用其进行毛细管电泳对于遗传性疾病的诊断将更加快速、准确、简便、灵敏.     

Characterization of anacardic acids by micellar electrokinetic chromatography and mass spectrometry

A possibility of using capillary electrophoresis for separation of anacardic acids (6-alkylsalicylic acids) has been studied. Conventional micellar electrokinetic chromatography (MEKC) in non-coated fused silica capillaries and reversed-flow micellar electrokinetic chromatography (RF-MEKC) in capillaries coated with polydimethylacrylamide was applied for separation of anacardic acids extracted from cashew nuts. Influence of the composition of background electrolyte on the resolution of anacardic acid isomers was evaluated. Separations were performed using sodium dodecyl sulphate (SDS) micelles and mixed micelles of SDS and polyoxyethylene lauryl ether as a pseudostationary phase. To further improve the separation in RF-MEKC, beta-cyclodextrin and a dual cyclodextrin system of beta-cyclodextrin with heptakis-6-sulphato-beta-cyclodextrin was added to the working electrolyte. Best separation of anacardic acids were achieved in the polydimethylacrylamide-coated capillary using 10 mM phosphate background electrolyte pH 6.5 with addition of 1 M urea, 20% acetonitrile, 10 mM of beta-cyclodextrin and 1 mM of heptakis-6-sulfo-beta-cyclodextrin. Mass spectrometry was used for the identification of anacardic acids in the extract from cashew nuts in single and tandem mode using Q-TOF instrument. Nine anacardic acids were identified in the extract form the cashew nuts.