多糖凝胶的孔径和化学结构对单壁碳纳米管流动性和选择性分离的影响

基于凝胶柱色谱分离技术研究了单分散的单壁碳纳米管(SWCNTs)在不同化学结构多孔多糖凝胶中的流动特性以及对金属型(m-)/半导体型(s-)SWCNTs分离的影响.通过比较SWCNTs在一系列不同孔径的葡聚糖Sephacryl凝胶中的流动行为,发现减小孔径尺寸能够增强s-SWCNTs与凝胶之间的吸附作用力,使大直径的m-SWCNTs快速地流过凝胶颗粒,而选择性地保留了小直径的s-SWCNTs.进一步发现多糖凝胶化学结构比孔径尺寸在SWCNTs的m/s分离中起着更重要的作用.当基于葡聚糖结构的Sephacryl凝胶中的氨基结构被琼脂糖结构所取代时,如Superdex 200和Sepharose 2B凝胶会增强它们与SWCNTs之间的作用力,使SWCNTs的保留时间延长,降低了s-SWCNTs的选择性和纯度.此外,即使拥有与Sephacryl S100类似的孔径范围,当Sephacryl凝胶中的氨基被疏水环氧丙烷基团取代时,葡聚糖凝胶Sephadex G100与SWCNTs的作用力很弱,导致所有SWCNTs快速流动,无法实现SWCNTs的m/s分离.因而,我们认为凝胶孔径和化学结构共同影响并调控了SWCNTs的m/s分离的选择性、纯度以及分离效率.     

多糖凝胶的孔径和化学结构对单壁碳纳米管流动性和选择性分离的影响

基于凝胶柱色谱分离技术研究了单分散的单壁碳纳米管（SWCNTs）在不同化学结构多孔多糖凝胶中的流动特性以及对金属型（m-）/半导体型（s-）SWCNTs 分离的影响. 通过比较SWCNTs 在一系列不同孔径的葡聚糖Sephacryl 凝胶中的流动行为,发现减小孔径尺寸能够增强s-SWCNTs 与凝胶之间的吸附作用力,使大直径的m-SWCNTs 快速地流过凝胶颗粒,而选择性地保留了小直径的s-SWCNTs. 进一步发现多糖凝胶化学结构比孔径尺寸在SWCNTs 的m/s 分离中起着更重要的作用. 当基于葡聚糖结构的Sephacryl 凝胶中的氨基结构被琼脂糖结构所取代时,如Superdex 200 和Sepharose 2B凝胶会增强它们与SWCNTs 之间的作用力,使SWCNTs 的保留时间延长,降低了s-SWCNTs 的选择性和纯度. 此外,即使拥有与Sephacryl S100类似的孔径范围,当Sephacryl 凝胶中的氨基被疏水环氧丙烷基团取代时,葡聚糖凝胶Sephadex G100 与SWCNTs 的作用力很弱,导致所有SWCNTs 快速流动,无法实现SWCNTs 的m/s 分离. 因而,我们认为凝胶孔径和化学结构共同影响并调控了SWCNTs的m/s分离的选择性、纯度以及分离效率.     

单壁碳纳米管在胆酸类表面活性剂中的分散及手性碳管(6,5)的双水相分离方法

选用牛磺脱氧胆酸钠和脱氧胆酸钠等6种胆酸类表面活性剂,考察其对单壁碳纳米管(SWCNTs)的分散能力.紫外-可见-近红外吸收光谱测试结果表明,在超声功率为225 W,超声时间1 h的分散条件下,胆酸类表面活性剂均能对SWCNTs均匀分散,均可作为SWCNTs的分散剂.在相同条件下,质量分数为2%的牛磺脱氧胆酸钠对SWCNTs的分散能力最强,脱氧胆酸钠对SWCNTs的分散能力最弱.此外,还采用聚乙二醇/葡聚糖双水相系统对SWCNTs分散液进行了萃取分离,获得了纯度较高的手性SWCNTs(6,5).所筛选的SWCNTs分散剂及采用的双水相分离方法为单一手性SWCNTs的分离提供了一定的技术参考.     

琼脂糖性质对凝胶电泳法分离金属性和半导体性单壁碳纳米管的影响

利用琼脂糖凝胶电泳分离单壁碳纳米管(SWCNTs)技术, 考察了MB, Agarose, Agarose B和LRU 4种琼脂糖对SWCNTs分离效率的影响. 紫外-可见-近红外(UV-Vis-NIR)吸收光谱研究结果表明, 不同的琼脂糖对SWCNTs中s-SWCNTs的分离效率影响较小, 而对m-SWCNTs的分离效率影响较大. 分析4种琼脂糖凝胶的凝胶强度和凝胶网孔尺寸等发现, 影响SWCNTs中m-SWCNTs分离效率的主要因素是琼脂糖的凝胶强度和琼脂糖凝胶形成的网孔尺寸, 小的凝胶网孔尺寸有利于m-SWCNTs富集, 高凝胶强度则不利于其富集.     

超声分散对单壁碳纳米管分离的影响

利用凝胶柱色谱技术, 研究者们通过两步或多步淋洗的方法实现了不同导电属性或电子结构单壁碳纳米管(SWCNTs)的分离, 并提出其分离机制主要是由不同导电属性和电子结构的SWCNTs 与凝胶填料之间作用力的差异所导致的. 基于凝胶柱色谱分离技术, 本文重点考察了超声时间对单壁碳纳米管单分散以及金属型/半导体型SWCNTs 分离的影响. 在一定的低超声功率下, 适当增加超声时间有利于SWCNTs 在十二烷基硫酸钠(SDS)溶液中的单分散. 紫外-可见-近红外(UV-Vis-NIR)吸收光谱、拉曼(Raman)光谱和荧光(PL)光谱表征结果表明, 2 h的超声条件是获得高纯度的金属型以及不同直径分布的半导体型SWCNTs 的最优条件. 我们认为不同超声时间对SWCNTs 分离的影响主要是改变了SWCNTs 的单分散性和长度, 调制了不同SWCNTs 与凝胶之间作用力的差异, 从而导致了不同SWCNTs分离结果.     

Structural analysis of protein complexes with sodium alkyl sulfates by small-angle scattering and polyacrylamide gel electrophoresis

Small-angle X-ray (SAXS) and neutron (SANS) scattering is used to probe the structure of protein-surfactant complexes in solution and to correlate this information with their performance in gel electrophoresis. Proteins with sizes between 6.5 to 116 kDa are denatured with sodium alkyl sulfates (SC(x)S) of variable tail lengths. Several combinations of proteins and surfactants are analyzed to measure micelle radii, the distance between micelles, the extension of the complex, the radius of gyration, and the electrophoretic mobility. The structural characterization shows that most protein-surfactant complexes can be accurately described as pearl-necklace structures with spherical micelles. However, protein complexes with short surfactants (SC(8)S) bind with micelles that deviate significantly from spherical shape. Sodium decyl (SC(10)S) and dodecyl (SC(12)S, more commonly abbreviated as SDS) sulfates result in the best protein separations in standard gel electrophoresis. Particularly, SC(10)S shows higher resolutions for complexes of low molecular weight. The systematic characterization of alkyl sulfate surfactants demonstrates that changes in the chain architecture can significantly affect electrophoretic migration so that protein-surfactant structures could be optimized for high resolution protein separations.     

An inexpensive microslab gel DNA electrophoresis system with real-time fluorescence detection

In this paper, we describe the construction of a simple yet powerful gel electrophoresis apparatus that can be used to perform size-selective separations of DNA fragments in virtually any laboratory. This system employs a microslab gel format with a novel gel casting technique that eliminates the need for delicate combs to define sample loading wells. The compact size of the microslab gel format allows rapid separations to be performed at low voltages using submicroliter sample volumes. Real time fluorescence detection of the migrating DNA fragments is accomplished using an inexpensive digital microscope that directly connects to any PC with a USB interface. The microscope is readily adaptable for this application by replacing its white light source with a blue light-emitting diode (LED) and adding an appropriate emission filter. Both polyacrylamide and agarose gels can be used as separation matrices. Separation performance was characterized using standard dsDNA ladders, and correct sizing of a 191 bp PCR product was achieved in 15 min. The low cost and simplicity of this system makes it ideally suited for use in a variety of laboratory and educational settings.     

A versatile microfabricated platform for electrophoresis of double- and single-stranded DNA

We demonstrate a versatile microfabricated electrophoresis platform, incorporating arrays of integrated on-chip electrodes, heaters, and temperature sensors. This design allows a range of different sieving gels to be used within the same device to perform separations involving both single- and double-stranded DNA over distances on the order of 1 cm. We use this device to compare linear and cross-linked polyacrylamide, agarose, and thermo-reversible Pluronic-F127 gels on the basis of gel casting ease, reusability, and overall separation performance using a 100 base pair double-stranded DNA ladder as a standard sample. While cross-linked polyacrylamide matrices provide consistently high-quality separations in our system over a wide range of DNA fragment sizes, Pluronic gels also offer compelling advantages in terms of the ability to remove and reload the gel. Agarose gels offer good separation performance, however, additional care must be exercised to ensure consistent gel properties as a consequence of the need for elevated gel loading temperatures. We also demonstrate the use of denaturing cross-linked polyacrylamide gels at concentrations up to 19% to separate single-stranded DNA fragments ranging in size from 18 to 400 bases in length. Primers differing by 4 bases at a read length of 30 bases can be separated with a resolution of 0.9-1.0 in under 20 min. This level of performance is sufficient to conduct a variety of genotyping assays including the rapid detection of single nucleotide polymorphisms (SNPs) in a microfabricated platform. The ability to use a single microelectrophoresis system to satisfy a wide range of separation applications offers molecular biologists an unprecedented level of flexibility in a portable and inexpensive format.     

A simple capillary gel electrophoresis approach for efficient and reproducible DNA separations. Analysis of genetically modified soy and maize

It is generally assumed that in order to achieve suitable separations of DNA fragments, capillary gel electrophoresis (CGE)-coated capillaries should be used. In this work, a new method is presented that allows to obtain reproducible CGE separations of DNA fragments using bare fused-silica capillaries without any previous coating step. The proposed method only requires: (i) a capillary washing with 0.1 M hydrochloric acid between injections and (ii) a running buffer composed of Tris-phosphate-ethylenediamine tetraacetic acid (EDTA) and 4.5% of 2-hydroxyethyl cellulose (HEC) as sieving polymer. The use of this new CGE procedure gives highly resolved and reproducible separations of DNA fragments ranging from 50 to 750 bp. The separation of these DNA fragments is accomplished in less than 30 min with efficiencies up to 1.7 x 10(6) plates/m. Reproducibility values of migration times (given as %RSD) for the analyzed DNA fragments are better than 1.0% (n = 4) for the same day, 2.2% (n = 16) for four different days, and 2.3% (n = 16) for four different capillaries. The usefulness of this separation method is demonstrated by detecting genetically modified maize and genetically modified soy after DNA amplification by PCR. This new CGE procedure together with LIF as detector provides sensitive analysis of 0.9% of Bt11 maize, Mon810 maize, and Roundup Ready soy in flours with S/ N up to 542. These results demonstrate the usefulness of this procedure to fulfill the European regulation on detection of genetically modified organisms in foods.     

Fast separation of single-stranded oligonucleotides by capillary electrophoresis using OliGreen as fluorescence inducing agent

The fast separation of oligonucleotide (oligos) sizing marker by CE using OliGreen and including effects due to the concentration of separation medium and urea denaturant is presented. OliGreen dye is found to be more sensitive than ethidium bromide (by a factor of about 6 based on S/N considerations) for the oligos' separations. Higher concentration of F127 in 1xTris-boricacid-EDTA (TBE) up to 30% w/v leads to better resolution of oligos separations. The addition of urea into the separation medium decreases the sensitivity. With an optimized running condition, the oligos sizing marker could be successfully separated with 1-base resolution within 1.3 min by using 30% w/v F127/1xTBE solution as the separation medium at an applied electric field of 800 V/cm in a 3 cm long capillary, the fastest capillary gel electrophoresis separation with high resolution reported to date for oligos in the similar size range.     

Aqueous multiphase systems of polymers and surfactants provide self-assembling step-gradients in density

This Communication demonstrates the generation of over 300 phase-separated systems-ranging from two to six phases-from mixtures of aqueous solutions of polymers and surfactants. These aqueous multiphase systems (MuPSs) form self-assembling, thermodynamically stable step-gradients in density using a common solvent, water. The steps in density between phases of a MuPS can be very small (Δρ ≈ 0.001 g/cm(3)), do not change over time, and can be tuned by the addition of co-solutes. We use two sets of similar objects, glass beads and pellets of different formulations of Nylon, to demonstrate the ability of MuPSs to separate mixtures of objects by differences in density. The stable interfaces between phases facilitate the convenient collection of species after separation. These results suggest that the stable, sharp step-gradients in density provided by MuPSs can enable new classes of fractionations and separations based on density.     

Enhanced adsorption affinity of anionic perylene-based surfactants towards smaller-diameter SWCNTs

We present evidence from multiple characterization methods, such as emission spectroscopy, zeta potential, and analytical ultracentrifugation, to shed light on the adsorption behavior of synthesized perylene surfactants on single-walled carbon nanotubes (SWCNTs). On comparing dispersions of smaller-diameter SWCNTs prepared by using cobalt-molybdenum catalysis (CoMoCAT) with the larger-diameter SWCNTs prepared by high-pressure carbon monoxide decomposition (HiPco), we find that the CoMoCAT-perylene surfactant dispersions are characterized by more negative zeta potentials, and higher anhydrous specific volumes (the latter determined from the sedimentation coefficients by analytical ultracentrifugation), which indicates an increased packing density of the perylene surfactants on nanotubes of smaller diameter. This conclusion is further supported by the subsequent replacement of the perylene derivatives from the nanotube sidewall by sodium dodecyl benzene sulfonate (SDBS), which first occurs on the larger-diameter nanotubes. The enhanced adsorption affinity of the perylene surfactants towards smaller-diameter SWCNTs can be understood in terms of a change in the supramolecular arrangement of the perylene derivatives on the scaffold of the SWCNTs. These findings represent a significant step forward in understanding the noncovalent interaction of π-surfactants with carbon nanotubes, which will enable the design of novel surfactants with enhanced selectivity for certain nanotube species.     

Separation and purification of oligonucleotides using a new bonded-phase packing material

We describe a new bonded-phase packing material, based upon surface-stabilised microparticulate silica, suitable for the rapid separation and purification of oligonucleotides. Columns packed with this material were demonstrated to give rapid separations of individual oligonucleotide species of up to 44 base units with high purity; agarose gel electrophoresis showed that the products were essentially single bands, with only trace quantities of the (n-1)-mer present. Baseline resolution of the desired oligomer from (n +/- 1)-mer was achieved under preparative loading conditions, where up to 200-300 micrograms of oligonucleotide could be separated. The separation was essentially independent of structure or sequence of the oligonucleotides. The retention mechanism of the oligonucleotides was investigated, and the results used to determine the optimum column configuration and separation conditions.     

Unraveling the 13C NMR chemical shifts in single-walled carbon nanotubes: dependence on diameter and electronic structure

The atomic specificity afforded by nuclear magnetic resonance (NMR) spectroscopy could enable detailed mechanistic information about single-walled carbon nanotube (SWCNT) functionalization as well as the noncovalent molecular interactions that dictate ground-state charge transfer and separation by electronic structure and diameter. However, to date, the polydispersity present in as-synthesized SWCNT populations has obscured the dependence of the SWCNT (13)C chemical shift on intrinsic parameters such as diameter and electronic structure, meaning that no information is gleaned for specific SWCNTs with unique chiral indices. In this article, we utilize a combination of (13)C labeling and density gradient ultracentrifugation (DGU) to produce an array of (13)C-labeled SWCNT populations with varying diameter, electronic structure, and chiral angle. We find that the SWCNT isotropic (13)C chemical shift decreases systematically with increasing diameter for semiconducting SWCNTs, in agreement with recent theoretical predictions that have heretofore gone unaddressed. Furthermore, we find that the (13)C chemical shifts for small diameter metallic and semiconducting SWCNTs differ significantly, and that the full-width of the isotropic peak for metallic SWCNTs is much larger than that of semiconducting nanotubes, irrespective of diameter.     

Separation of plasmid DNA isoforms by highly converging flow through small membrane pores

Plasmid DNA isoforms can be separated by both agarose gel electrophoresis and a variety of chromatographic methods, but both of these approaches have significant shortcomings in terms of scalability, throughput, and/or resolution. This study provides the first demonstration that the supercoiled, linear, and open-circular isoforms of plasmid DNA can be effectively separated based on differences in their elongational flexibility in the highly converging flow field that is established during membrane ultrafiltration. Data were obtained with plasmids from 3 to 17 kbp in size using commercially available cellulose ultrafiltration membranes with pores an order of magnitude smaller than the DNA root-mean-square radius of gyration. High-resolution separations were achieved by controlling the filtrate flux between the critical flux values required for transmission of the individual isoforms. The separation behavior in ultrafiltration was very different than that observed in size exclusion chromatography or agarose gel electrophoresis due to differences in the underlying separation mechanisms. The simplicity of the ultrafiltration process makes this approach attractive for a wide range of applications, including large-scale purification of plasmid DNA for gene therapy.     

Molecular perspective on diazonium adsorption for controllable functionalization of single-walled carbon nanotubes in aqueous surfactant solutions

Functionalization of single-walled carbon nanotubes (SWCNTs) using diazonium salts allows modification of their optical and electronic properties for a variety of applications, ranging from drug-delivery vehicles to molecular sensors. However, control of the functionalization process remains a challenge, requiring molecular-level understanding of the adsorption of diazonium ions onto heterogeneous, charge-mobile SWCNT surfaces, which are typically decorated with surfactants. In this paper, we combine molecular dynamics (MD) simulations, experiments, and equilibrium reaction modeling to understand and model the extent of diazonium functionalization of SWCNTs coated with various surfactants (sodium cholate, sodium dodecyl sulfate, and cetyl trimethylammonium bromide). We show that the free energy of diazonium adsorption, determined using simulations, can be used to rank surfactants in terms of the extent of functionalization attained following their adsorption on the nanotube surface. The difference in binding affinities between linear and rigid surfactants is attributed to the synergistic binding of the diazonium ion to the local "hot/cold spots" formed by the charged surfactant heads. A combined simulation-modeling framework is developed to provide guidance for controlling the various sensitive experimental conditions needed to achieve the desired extent of SWCNT functionalization.     

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2× or 4×48 base pair repeats) of the dopamine D₄ receptor gene (D₄DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.     

Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels

The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.     

Separation of transfer RNA and 5S ribosomal RNA using capillary electrophoresis

Capillary gel electrophoresis and capillary electrophoresis using entangled polymer solutions was investigated for their applicability for the separation of low-molecular-mass RNAs (transfer RNA and 5S ribosomal RNA), with a size range of 70–135 nucleotides, from bacteria. Cross-linked polyacrylamide gel-filled capillaries (3 and 5%) were used for capillary gel electrophoresis. Good resolution was obtained suing gel-filled capillaries only for small tRNAs with lengths to 79 nucleotides, larger tRNAs and 5S rRNA could not be resolved using this method. Buffers containing sieving additives were employed to improve separations of RNA by capillary electrophoresis using entangled polymer solutions. The use of linear sieving polymers in buffers resolved 5S rRNA and tRNAs, even when they possessed only different secondary structure or small differences in length (1–5 nucleotides).     

Capillary electrophoresis of covalently functionalized single‐chirality carbon nanotubes

We demonstrate the separation of chirality‐enriched single‐walled carbon nanotubes (SWCNTs) by degree of surface functionalization using high‐performance CE. Controlled amounts of negatively charged and positively charged functional groups were attached to the sidewall of chirality‐enriched SWCNTs through covalent functionalization using 4‐carboxybenzenediazonium tetrafluoroborate or 4‐diazo‐N,N‐diethylaniline tetrafluoroborate, respectively. Surfactant‐ and pH‐dependent studies confirmed that under conditions that minimized ionic screening effects, separation of these functionalized SWCNTs was strongly dependent on the surface charge density introduced through covalent surface chemistry. For both heterogeneous mixtures and single‐chirality‐enriched samples, covalently functionalized SWCNTs showed substantially increased peak width in electropherogram spectra compared to nonfunctionalized SWCNTs, which can be attributed to a distribution of surface charges along the functionalized nanotubes. Successful separation of functionalized single‐chirality SWCNTs by functional density was confirmed with UV‐Vis‐NIR absorption and Raman scattering spectroscopies of fraction collected samples. These results suggest a high degree of structural heterogeneity in covalently functionalized SWCNTs, even for chirality‐enriched samples, and show the feasibility of applying CE for high‐performance separation of nanomaterials based on differences in surface functional density.