Preparation of various silica-based columns for capillary electrochromatography by in-column derivatization

Chemically bonded silica gels were prepared in a capillary by pumping an ethanolic solution of a silylating reagent, such as octadecyltrimethoxysilane, 3-aminopropyltrimethoxysilane and dimethyloctadecyltrimethoxysilylpropylammonium chloride into a heated capillary packed with bare silica particles. The silylation reactions were completed in a short time and thus-prepared columns showed high column efficiency and high reproducibility. Examples are shown for the separation of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of aldopentoses on a 3-aminopropylated silica column and benzoate homologues as well as PMP derivatives of the component monosaccharides of glycoproteins on an octadecylammonium column. Since the presence of frit filters hampers high efficiency separation, an attempt was made to fix the bed of modified silica gel particles to the capillary inner wall by a cross-linking technique. The results indicated that this technique is promising.     

Miniaturization in carbohydrate analysis

Recent progress of microchip electrophoresis (ME) of carbohydrates is overviewed. Carbohydrate analysis by ME encounters difficulties such as lack of electric charge and deficiency of a chromophore/fluorophore in analyte molecules, however, it benefits from the accumulated knowledge of capillary electrophoresis (CE) and rapid separation of simple sugars also by ME, with high column efficiency comparable to CE, has become possible. Analysis at high pH, with electrochemical detection, is a promising approach because carbohydrates can be ionized by weak dissociation of the hydroxyl groups and the in situ formed ionic species can be effectively separated by the zone electrophoresis mode. The separated species can be sensitively monitored by electrochemical detection on a gold or copper electrode. Ionization as borate complexes and refractometric detection is also possible, though sensitivity is lower. Introduction of UV-absorbing or fluorescent tags is potentially useful but the time-consuming derivatization processes sacrifice the rapidity of ME. Examples of ME of carbohydrates as 1-phenyl-3-methyl-5-pyrazolone (PMP; for simple mono- and oligosaccharides with UV detection), 8-aminopyrene-1,3,6-trisulfonate (APTS; for oligosaccharides ladders with LIF detection), and 4-nitro-2,1,3-benzoxadiazole (NBD-F; for amino sugars and aminoalditols with LIF detection) derivatives are presented, with details of the analytical conditions. Since ME in a short separation channel enables rapid analysis within 1 min, it presents an ideal tool for clinical analysis, as shown in a few papers reporting protocols for specific blood glucose assay. Finally, the usefulness of microfluidic reactors and microarrays for enzyme-assisted carbohydrate analysis as well as glycan profiling is pointed out.     

柱前衍生化高效液相色谱法分析多糖中的单糖组成

报道了多糖中单糖组成的柱前衍生化高效液相色谱测定方法。采用反向高效液相色谱250nm紫外检测和使用梯度洗脱，6种还原单糖的1－苯基－3－甲基－5－吡唑啉酮衍生实现了良好的分离并具有良好的峰形。对单糖组成的定量测定进行了方法学考察，建立了单糖组成分析的数据分析方法，并用所建立方法对一个多糖中的单糖组成进行了分析，获得良好的重复性。     

Chiral separation of NBD-amino acids by ligand-exchange micro-channel electrophoresis.

The chiral separation of amino acid derivatives by ligand-exchange electrophoresis in a microchannel chip was performed for the first time. A Cu(II) complex with L-prolinamide was used as a chiral selector. The migration behaviors of eleven NBD-DL-amino acids were investigated by ligand-exchange capillary electrophoresis (LE-CE). The enantiomer of five NBD-amino acids (Ser, Thr, Val, Phe and His) could be separated by LE-CE using a 20 mM ammonium acetate buffer (pH 9.0) containing 10 mM copper acetate, 20 mM L-prolinamide and 1 mM SDS. NBD-His was eluted in the order D-form and L-form, while the elution order of another enantiomers was L-form and D-form. Under this condition, the enantioseparation of these five NBD-amino acids by ligand-exchange microchip electrophoresis (LE-ME) was investigated using a glass microchip. The enantioseparation of NBD-Ser, -Thr and -His could be successfully accomplished by LE-ME. LE-ME was superior to LE-CE in terms of the short migration time and a good enantiomeric separation.     

Investigation of different combinations of derivatization, separation methods and electrospray ionization mass spectrometry for standard oligosaccharides and glycans from ovalbumin

Derivatization procedures using 1-phenyl-3-methyl-5-pyrazolone (PMP) and 2-aminonaphthalene trisulfone (ANTS) were selected among a number of well known methods for labelling carbohydrates. PMP derivatives were selected owing to our laboratory's previous high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) experience with these, whereas the ANTS-labelled compounds were prepared for fluorophore-assisted carbohydrate electrophoresis (FACE) separation. ANTS-oligosaccharide standards were characterized to study their ionization patterns. Reversed-phase and normal-phase HPLC systems were coupled on-line with ESI-MS. Each necessitated its own mobile phase system which, in turn, imposed some important changes in the ionization conditions used and/or on the ionization patterns and spectra obtained. Following characterization of the intact glycoprotein ovalbumin with ESI-MS, its glycans were detached using the enzyme PNGase-F. The glycans were subjected to PMP and ANTS derivatization. It was very difficult to separate ANTS derivatives by reversed-phase HPLC owing to lack of retention, and normal-phase HPLC offered reasonable retention with limited separation. PMP compounds overall yielded better normal- and reversed-phase separations and improved sensitivity over the ANTS-labelled sugars, for which negative mode ESI had to be used. The combination of ESI of intact ovalbumin and ESI of PMP-glycans gave rise to the detection of over 20 different glycoforms, excluding the possible presence of structural isomers for each sugar composition detected.     

Frontal analysis on a microchip

Conventional microchip applications involving capillary electrophoresis (CE) typically inject a sample along one channel and use an intersection of two channels to define the sample plug--the portion of sample to be analysed along a second channel. In contrast to this method of zone separation, frontal analysis proceeds by injecting sample continuously into a single channel or column. Frontal analysis is more common in macroscopic procedures but there are benefits in sensitivity and device density to its application to electrophoresis on microchips. This work compares conventional microchip zone analysis with frontal analysis in the separation of PCR products. Although we detect on the order of 5000 fluorophores with a compact instrument using the zone separation CE method, we found a several-fold increase in the effective signal-to-noise ratio by using a frontal analysis method. By removing the need for additional channels and reservoirs the frontal method would allow device densities to be significantly increased, potentially improving the cost-effectiveness of microchip analyses in applications such as medical diagnostics.     

Property of ionic liquid in electrophoresis and its application in chiral separation on microchips

The aqueous solution of a kind of room-temperature ionic liquids (RTILs), 1-ethyl-3-methylimidazolium-tetrafluoroborate (1E-3MI-TFB), demonstrated its exclusive electroosmotic property in microchip electrophoresis. It was applied as the working electrolyte for chiral separation in glass microchip electrophoresis. Compared with boric acid buffer, 1E-3MI-TFB aqueous solution exhibited a broader separation window for enantiomers of dipeptides. Then the influences of chiral selector, pH and concentration on efficiency of chiral separation were discussed in detail. The unique mechanism of the generation of EOF was explored in a glass microchannel using 1E-3MI-TFB aqueous solution as working electrolyte. A possible status of 1E-3MI cation in water was suggested at the first time, which facilitated the explanation of EOF and its characteristics in glass microchannel. Additionally, microchip electrophoresis using 1E-3MI-TFB aqueous solution was successfully applied to the chiral separation of complex enantiomers of dipeptides. RTILs aqueous solution, as the electrolyte for the separation of complicated optical isomers, could lead to a revolution in the analytical methods of chiral or conformational analysis for biomolecules.     

Study of a novel sample injection method (floating electrokinetic supercharging) for high-performance microchip electrophoresis of DNA fragments

Aiming to achieve high-performance analysis of DNA fragments using microchip electrophoresis, we developed a novel sample injection method, which was given the name of floating electrokinetic supercharging (FEKS). In the method, electrokinetic injection (EKI) and ITP preconcentration of samples was performed in a separation channel, connecting two reservoir ports (P3 and P4) on a cross-geometry microchip. At these two stages, side channels, crossing the separation channel, and their ports (P1 and P2) were electrically floated. After the ITP-stacked zones passed the cross-part, they were eluted for detection by using leading ions from P1 and P2 that enabled electrophoresis mode changing rapidly from ITP to zone electrophoresis (ZE). Possible sample leakage at the cross-part toward P1 and P2 was studied in detail on the basis of computer simulation using a CFD-ACE+ software and real experiments, through which it was validated that the analyte recovery to the separation channel was almost complete. The FEKS method successfully contributed to higher resolution and shorter analysis time of DNA fragments on the cross-microchip owing to more rapid switching from ITP status to ZE separation in comparison with our previous EKS procedure realized on a single-channel microchip. Without any degradation of resolution, the achieved LODs were on average ten times better than using conventional pinched injection.     

Influence of the pH on separating DNA by high-speed microchip electrophoresis.

Various factors are critical in resolving DNA molecules at high speed, including the separation medium, concentration, composition, and pH of the buffer, as well as the electric field strength. To this study, considered the composition of a buffer and the difference in the pH, while paying attention to whether the separation ability changes in the microchip electrophoresis of DNA. DNA separation was particularly affected by both the buffer composition and the pH. Under the optimal microchip electrophoresis conditions that were determined in this study, an improved resolution of a wider range of DNA fragment sizes was achieved. Moreover, the total separation time decreased from 240 s to 100 s. Thus, by simplifying and improving the DNA electrophoresis in the microchip, this technique is now widely applicable to several different scientific fields.     

Direct measurement of lithium in whole blood using microchip capillary electrophoresis with integrated conductivity detection

The direct measurement of lithium in whole blood is described. Using microchip capillary electrophoresis (CE) with defined sample loading and applying the principles of column coupling, alkali metals were determined in a drop of whole blood. Blood collected from a finger stick was mixed with anticoagulant and transferred onto the chip without extraction or removal of components. The electrokinetic transport of red blood cells inside the channels was studied to find sample loading conditions suitable for the analysis of lithium without injecting cells into the separation channel. Both bare glass chips and chips coated with polyacrylamide were used showing the behavior of the cells under different electroosmotic flow conditions. In serum a detection limit for lithium of 0.4 mmol/L was reached. Proteins quickly contaminated untreated chip surfaces but devices with coating gave reproducible electropherograms. In addition, potassium and sodium were also detected in the same separation run. To our knowledge, this is the first device to directly measure ions in whole blood with the use of capillary zone electrophoresis on a microchip.     

Integration of on-column immobilized enzyme reactor in microchip electrophoresis

A simple method integrating an immobilized enzyme reactor into a microchip electrophoresis device was developed. The enzyme immobilization into a microchip was performed by spotting and drying a drop of dissolved nitrocellulose (NC) on a glass substrate, and adsorbing enzyme on the reconstituted NC membrane. This enzyme-immobilized glass plate was assembled with a polydimethylsiloxane substrate on which the separation channel was fabricated. The advantage of this method is the ability to easily change the position and size of the reactor within the microchip electrophoresis device. A beta-galactosidase reaction was demonstrated with fluorescein di-beta-D-galactopyranoside using this integrated on-column enzyme reactor. A successful electrophoretic separation of its hydrolysis products, i.e., fluorescein mono-beta-D-galactopyranoside (FMG) and fluorescein, was achieved. Enzyme kinetics and inhibition of the beta-galactosidase using FMG and 2-phenylethyl beta-D-thiogalactoside, respectively, were also studied with microchip electrophoresis.     

Capillary electrophoresis microchip coupled with on-line chemiluminescence detection

In the present work, chemiluminescence detection was integrated with capillary electrophoresis microchip. The microchip was designed on the principle of flow-injection chemiluminescence system and capillary electrophoresis. It has three main channels, five reservoirs and a detection cell. As model samples, dopamine and catechol were separated and detected using a permanganate chemiluminescent system on the prepared microchip. The samples were electrokinetically injected into the double-T cross section, separated in the separation channel, and then oxidized by chemiluminescent reagent delivered by a home-made micropump to produce light in the detection cell. The electroosmotic flow could be smoothly coupled with the micropump flow. The detection limits for dopamine and catechol were 20.0 and 10.0 μM, respectively. Successful separation and detection of dopamine and catechol demonstrated the distinct advantages of integration of chemiluminescent detection on a microchip for rapid and sensitive analysis.     

High-sensitivity capillary gel electrophoretic analysis of DNA fragments on an electrophoresis microchip using electrokinetic injection with transient isotachophoretic preconcentration

The research adopted a single-channel microchip as the probe, and focused electrokinetic injection combined with transient isotachophoresis preconcentration technique on capillary electrophoresis microchip to improve the analytical sensitivity of DNA fragments. The channel length, channel width and channel depth of the used microchip were 40.5 mm, and 110 and 50 μm, respectively. The separation was detected by CCD (charge-coupled device) (effective LENGTH=25 mm, 260 nm). A 1/100 diluted sample (0.2 mg/l of each DNA fragment) of commercially available stepladder DNA sample could be baseline separated in 120 s with S/N=2–5. Compared with conventional chip gel electrophoresis, the proposed method is ideally suited to improve the sensitivity of DNA analysis by chip electrophoresis.     

Miniaturized two-dimensional capillary electrophoresis on a microchip for analysis of the tryptic digest of proteins

A two-dimensional capillary electrophoresis platform, combining isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), was established on a microchip with the channel width and depth as 100 mum and 40 mum, respectively. With polyacrylamide as permanent coating, EOF in the microchannel, which could impair the separation, was decreased to 3.4x10(-9)m(2).V(-1).s(-1), about 1/10 of that obtained in the uncoated set-up. During the separation, peptides were first focused by IEF in the first dimensional channel, and then directly driven into the perpendicular channel by controlling the applied voltages, and separated by CZE. Effects of various experimental parameters, including the electric field strength, channel length, and injection frequency from the first to the second dimensional separation channel, were studied. Under optimized condition, the digests of BSA and proteins extracted from E. coli were separated, and a peak capacity of 540 was obtained, which was far greater than that obtained by each single dimensional separation. All these results showed the promise of multidimensional separation on a microchip for the high-throughput and high-resolution analysis of complex samples.     

微流体芯片电泳技术对人血清蛋白的快速分离

通过微流体芯片电泳技术分离人血清蛋白,探讨了常见十字形微流体芯片上样品的电动进样与分离过程,分析了在十字芯片上的进样时间和电压设置对后续样品检测和定量的影响。采用的缓冲体系为:100mmol/L H3BO3,50mmol/L NaCl,5%Dextran(以NaOH调至pH 8.3),该缓冲液能够有效分离人血清蛋白中的白蛋白(Albumin)和4种球蛋白(α1-,α2-,β-,和γ-globulin),并且给出了它们在该缓冲体系中的淌度估算范围为5.15×10-5～47.2×10-5 cm2/(V.s)。在芯片上2min之内可以完成进样和分离,相比于常用的毛细管区带电泳,提高了分析速度。     

Sensitive capillary electrophoresis microchip determination of trinitroaromatic explosives in nonaqueous electrolyte following solid phase extraction

A capillary electrophoresis (CE) microchip is utilized for the sensitive separation and detection of three trinitroaromatic explosives: 1,3,5-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), in the presence of 10 other explosives and explosive derivatives in nonaqueous electrolyte (acetonitrile/methanol 87.5/12.5 (v/v), 2.5 mM NaOH, 1 mM sodium dodecyl sulfate (SDS)). The chemical reaction of bases, e.g. hydroxide or methoxide ions, with trinitroaromatic compounds forms red colored derivatives that can be easily detected using a green light emitting diode (LED) on the microchip. Two surfactants bearing opposite charge, cetyltrimethylammonium bromide (CTAB) and SDS are compared with respect to their effect on separation times, detection limits and resolving powers for separating these explosives. All microchip separations were achieved in <20 s. In the absence of solid phase extraction (SPE), the detection limits obtained for the trinitroaromatic explosives were as follows: TNB, 60 μg/l; TNT, 160 μg/l and tetryl, 200 μg/l. By coupling the microchip separation with ex situ SPE, the detection limits for detecting these three explosives in seawater were lowered by 240 to more than 1000 times: TNB, 0.25 μg/l; TNT, 0.34 μg/l and tetryl, 0.19 μg/l.     

Dynamic coating using methylcellulose and polysorbate 20 for nondenaturing electrophoresis of proteins on plastic microchips

A dynamic coating using methylcellulose (MC) and a nonionic detergent (polysorbate 20) was developed, which controlled protein adsorption onto the surface of microchannels on a microchip made of poly(methyl methacrylate) (PMMA). Optimum concentration of polysorbate 20 in combination with the range of MC concentrations controlled the protein adsorption onto the microchannel surface, and increased the solubility of the protein samples while facilitating the injection of high concentrations of MC solutions into the microchannels. Higher concentrations of nonionic detergent increased the EOF mobility as opposed to the electrophoretic mobility and caused the electrophoresis to fail. Nondenaturing microchip electrophoresis of protein samples with molecular masses ranging from 20 to 100 kDa were completed in 100 s. Also, successful separation of a BSA sample and its complex with anti-BSA mAb ( 220 kDa) was achieved on a PMMA microchip. The separation exhibited high reproducibility in both migration time (RSD = 1%) and peak area (RSD = 10-15%).     

Nanoband electrode for high-performance in-channel amperometric detection in dual-channel microchip capillary electrophoresis

A nanoband electrode detector integrated with a dual-channel polydimethylsiloxane microchip is proposed for in-channel amperometric detection in microchip capillary electrophoresis. Gold nanoband electrodes, which were fabricated on SU-8 substrates with a 100-nm-width gold layer, were introduced into the dual-channel microchip to be an electrochemical detector. Due to the nano-sized width of the detector, the noise of the amperometric detection was significantly reduced, and a high separation resolution was achieved for monitoring the analytes. The detection sensitivity of the system was improved by high signal-to-noise ratio, and a low detection limit on microchip was obtained for p-aminophenol (2.09 nM). Because of the high resolution in measuring half-peak width, the plate number that is used to evaluate the separation efficiency was 1.5-fold higher than that using 50-μm-width electrochemical detector. The effect of sample injection time and data acquisition time on separation efficiency was investigated, and an attractive separation efficiency was achieved with a plate number up to 17,500.     

A highly efficient and versatile microchip capillary electrophoresis method for DNA separation using gold nanoparticle as a tag

A highly efficient and versatile method for DNA separation using Au nanoparticles (Au NPs) as a tag based on microchip capillary electrophoresis (MCE) was developed. The thiol-modified DNA-binding Au NPs were utilized as a tag. Target DNA was sandwiched between Au NPs and probe DNA labeled with horseradish peroxidase (HRP). In electrophoresis separation, the difference in electrophoretic mobility between free probe and probe-target complex was magnified by Au NPs, which enabled the resulting mixture to be separated with high efficiency by microchip capillary electrophoresis. Horseradish peroxidase was used as a catalytic label to achieve sensitive electrochemical DNA detection via fast catalytic reactions. With this protocol, 27-mer DNA fragments with different sequences were separated with high speed and high resolution. The proposed method was critical to achieve improved DNA separations in hybridization analyses.     

超高速平板通道毛细管电泳

超高速平板通道毛细管电泳是９０年代发展的一种秒级分离的新颖技术。应用现代微电子光刻技术将化学反应。进样、分离和检测等组合在数厘米玻片上。实现分离分析的小型化、集成化、一体化和自动化。