Label-free fluorescence detection in capillary and microchip electrophoresis

Herein, we summarize the current status of native fluorescence detection in microchannel electrophoresis, with a strong focus on chip-based systems. Fluorescence detection is a powerful technique with unsurpassed sensitivity down to the single-molecule level. Accordingly fluorescence detection is attractive in combination with miniaturised separation techniques. A drawback is, however, the need to derivatize most analytes prior to analysis. This can often be circumvented by utilising excitation light in the UV spectral range in order to excite intrinsic fluorescence. As sensitive absorbance detection is challenging in chip-based systems, deep-UV fluorescence detection is currently one of the most general optical detection techniques in microchip electrophoresis, which is especially attractive for the detection of unlabelled proteins. This review gives an overview of research on native fluorescence detection in capillary (CE) and microchip electrophoresis (MCE) between 1998 and 2008. It discusses material aspects of native fluorescence detection and the instrumentation used, with particular focus on the detector design. Newer developments, featured techniques, and their prospects in the future are also included. In the last section, applications in bioanalysis, drug determination, and environmental analysis are reviewed with regard to limits of detection.     

微流控芯片毛细管电泳在蛋白质分离分析中的应用研究进展

重点介绍了近年来国内外在微流控芯片毛细管电泳法用于蛋白质分离分析方面的研究进展。按照分离模式的不同,综述了各种应用于蛋白质分离的微流控芯片毛细管电泳系统,讨论了抑制芯片中的蛋白吸附的各种方法,并展望了芯片毛细管电泳系统在蛋白质分离领域的发展前景。引用文献47篇。     

A DNA sieving matrix with thermally tunable mesh size

We present a "proof-of-concept" study showing that a blend of thermo-responsive and nonthermo-responsive polymers can be used to create a DNA sieving matrix with a thermally tunable mesh size, or "dynamic porosity". Various blends of two well-studied sieving polymers for CE, including hydroxypropylcellulose (HPC), a thermo-responsive polymer, and hydroxyethylcellulose (HEC), a nonthermo-responsive polymer, were used to separate a double-stranded DNA restriction digest (Phi X174-HaeIII). HPC exhibits a volume-phase transition in aqueous solution which results in a collapse in polymer coil volume at approximately 39 degrees C. Utilizing a blend of HPC and HEC in a ratio of 1:5 by weight, we investigated the effects of changing mesh size on DNA separation, as controlled by temperature. High-resolution DNA separations were obtained with the blended matrix at temperatures ranging from 25 degrees C to 38 degrees C. We evaluated changes in the selectivity of DNA separation with increasing temperature for certain pairs of small and large fragments. A pure HEC (nonthermo-responsive) matrix was used over the same temperature range as a negative control. In the blended matrix, we observe a maximum in selectivity at approximately 31 degrees C for small DNA, while a significant increase in the selectivity of large-DNA separation occurs at approximately 36 degrees C as the polymer mesh "opens". We also demonstrate, through a temperature ramping experiment, that this matrix can be utilized to obtain high-resolution separation of both small and large DNA fragments simultaneously in a single CE run. Blended polymer matrices with "dynamic porosity" have the potential to provide enhanced genomic analysis by capillary array or microchip electrophoresis in microfluidic devices with advanced temperature control.     

Application of capillary electrophoresis for the early diagnosis of cancer

Early diagnosis is the key to the effective treatment of cancer. The detection of cancer biomarkers plays a critical role not only in cancer early diagnosis, but also in classification and staging tumor progression, or assessment prognosis and treatment response. Currently, various molecular diagnostic techniques have been developed for cancer biomarker studies, with many of the more effective approaches requiring a separation step before detection. Capillary electrophoresis (CE) can perform rapid and efficient separation with small samples, which is well-suited for analysis of both small- and macro- molecule biomarkers in complex samples. CE has different separation modes and can couple to different detectors into a variety of platforms, such as conducting studies on DNA/ RNA point mutation, protein misexpression, and metabolite abnormality. Similarly, microchip capillary electrophoresis (MCE) appears as a very important biomarker screening platform with the merits of high throughput, integration, and miniaturization, which makes it a promising clinical tool. By hyphenated different detectors, or integrated with immunoassay, PCR/LDR and related technologies, MCE can be constructed into diverse platforms used in genomics, proteomics, and metabolomics study for biomarkers discovery. The multiplex biomarker screening approach via CE- or MCE-based platforms is becoming a trend. This paper focuses on studies of cancer biomarkers via CE/MCE platforms, based on the studies published over the past 3 years. Some recent CE applications in the field of cancer study, such as cancer theranostics, are introduced.     

Micro- and nanofluidics for DNA analysis

Miniaturization to the micrometer and nanometer scale opens up the possibility to probe biology on a length scale where fundamental biological processes take place, such as the epigenetic and genetic control of single cells. To study single cells the necessary devices need to be integrated on a single chip; and, to access the relevant length scales, the devices need to be designed with feature sizes of a few nanometers up to several micrometers. We will give a few examples from the literature and from our own research in the field of miniaturized chip-based devices for DNA analysis, including dielectrophoresis for purification of DNA, artificial gel structures for rapid DNA separation, and nanofluidic channels for direct visualization of single DNA molecules.     

Disposable polyester-toner electrophoresis microchips for DNA analysis

Microchip electrophoresis has become a powerful tool for DNA separation, offering all of the advantages typically associated with miniaturized techniques: high speed, high resolution, ease of automation, and great versatility for both routine and research applications. Various substrate materials have been used to produce microchips for DNA separations, including conventional (glass, silicon, and quartz) and alternative (polymers) platforms. In this study, we perform DNA separation in a simple and low-cost polyester-toner (PeT)-based electrophoresis microchip. PeT devices were fabricated by a direct-printing process using a 600 dpi-resolution laser printer. DNA separations were performed on PeT chip with channels filled with polymer solutions (0.5% m/v hydroxyethylcellulose or hydroxypropylcellulose) at electric fields ranging from 100 to 300 V cm(-1). Separation of DNA fragments between 100 and 1000 bp, with good correlation of the size of DNA fragments and mobility, was achieved in this system. Although the mobility increased with increasing electric field, separations showed the same profile regardless of the electric field. The system provided good separation efficiency (215,000 plates per m for the 500 bp fragment) and the separation was completed in 4 min for 1000 bp fragment ladder. The cost of a given chip is approximately $0.15 and it takes less than 10 minutes to prepare a single device.     

Electrophoretic separations on microfluidic chips

This review presents a brief outline and novel developments of electrophoretic separation in microfluidic chips. Distinct characteristics of microchip electrophoresis (MCE) are discussed first, in which sample injection plug, joule heat, channel turn, surface adsorption and modification are introduced, and some successful strategies and recognized conclusions are also included. Important achievements of microfluidic electrophoresis separation in small molecules, DNA and protein are then summarized. This review is aimed at researchers, who are interested in MCE and want to adopt MCE as a functional unit in their integrated microsystems.     

Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection

We developed a method for the analysis of multiplexed double-stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single-column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A Phi X174HinfI and a pGEM DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe(4) (+)) as the only cation in the buffer.     

Monolithic integration of well-ordered nanoporous structures in the microfluidic channels for bioseparation

Gel electrophoresis and capillary gel electrophoresis are widely used for the separation of biomolecules. With increasing demand in the miniaturized devices such as lab-on-a-chip, it is necessary to integrate such a separation component into a chip format. Here, we describe a simple approach to fabricate robust three-dimensional periodic porous nanostructures inside the microchannels for the separation of DNA molecules. In our approach, the colloidal crystals were first grown inside the microchannel using evaporation assisted self-assembly process. Then the void spaces among the colloidal crystals were filled with epoxy-based negative tone photoresist (SU-8). UV radiation was used to cure the photoresist at the desired area inside the microchannel. After subsequent development and nanoparticle removal, the well-ordered nanoporous structures inside the microchannel were obtained. Our results indicated that it was possible to construct periodic porous nanostructures inside the microchannels with cavity size around 300 nm and interconnecting pores around 30 nm. The mobility of large DNA molecules with different sizes was measured as a function of the applied electric field in the nanoporous materials. It was also demonstrated that 1 kilo-base pair (kbp) DNA ladders could be separated in such an integrated system within 10 min under moderate electric field.     

Cover Picture: Electrophoresis 2'2011

Issue no. 2 is a regular issue assembled of 16 solid and original research articles distributed over 3 distinct parts. Part I is on novel trends in fundamentals and methodologies including theoretical models for selectivity of charged solutes in MEKC, system peaks in indirect detection, measuring epimerization constants by MEEKC, bundled CE using micro‐structured fibers, 2‐D separations by coupling CIEF and CEC, high speed DNA CE, MCE of N‐glycans and mucin expression in a microfluidic gradient device. Part II is concerned with detection, sensitivity enhancement, on‐column preconcentration and microdialysis sampling involving the design of continuous full filling CEC‐ESI‐MS using nanoparticles, CE‐fluorescence using tapered optical fiber, CZE separation of pesticide residues in water samples with acid‐assisted on‐column preconcentration and CE‐LIF to detect neurotransmitter amino acids and carbamathione in brain microdialysis samples. Novel methods for the separation and profiling of various proteins and large nucleic fragments are described in 4 consecutive papers grouped in part III. Featured articles include: Theoretical models of separation selectivity for charged compounds in micellar electrokinetic chromatography (( 10.1002/elps.201000405 )) Bundled capillary electrophoresis using microstructured fibres ( 10.1002/elps.201000442 )) Two‐dimensional separation system by on‐line hyphenation of capillary isoelectric focusing with pressurized capillary electrochromatography for peptide and protein mapping ( 10.1002/elps.201000419 )) Microchip electrophoresis of N‐glycans on serpentine separation channels with asymmetrically tapered turns ( 10.1002/elps.201000461 ))     

Laser‐Induced Fluorometry for Capillary Electrophoresis

Laser‐induced fluorometry (LIF) has achieved the detection of single molecules, which ranks it among the most sensitive of detection techniques, whereas capillary electrophoresis (CE) is known as a powerful separation method with resolution that is beyond the reach of many other types of chromatography. Therefore, a coupling of LIF with CE has established an unrivaled analytical technique in terms of sensitivity and resolution. CE‐LIF has demonstrated excellent performance in bioanalytical chemistry for the high‐resolution separation and highly sensitive detection of DNAs, proteins, and small bioactive molecules. This review describes the CE‐LIF methods developed by the author's group that include indirect and direct detection using diode lasers, post‐column derivatization, and Hadamard transformation, as well as applications to the binding assays of specific DNA immunoassays of proteins and to the determination of anticancer drugs.     

毛细管电泳无胶筛分介质分离DNA的机理

快速、高效而灵敏的分离技术对于DNA的分析是至关重要的。使用无胶筛分介质的毛细管电泳是最重要的DNA分离技术之一,通常使用无交联的高分子溶液作为无胶筛分介质。本文在介绍高分子溶液理论的基础上,综述了DNA在毛细管电泳无胶筛分介质(缠结溶液和稀溶液)中的分离机理,主要包括Ogston筛分模型、各种修正的爬行模型、瞬态缠结偶合机理及其改进机理等。     

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.     

Analysis of glycans derived from glycoconjugates by capillary electrophoresis-mass spectrometry

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful MS and MS/MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high-order MS/MS (MS(n) ). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion-exchange chromatography, hydrophilic interaction chromatography and GC. However, CE and microfluidics CE (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to MS.     

Polymer solutions as a pseudostationary phase for capillary electrochromatographic separation of DNA diastereomers

The solutions of linear polymers traditionally used for DNA separation have been employed for the capillary electrophoresis (CE) of diastereomers of chemically modified DNA. The selectivity of diastereomeric separation of the phosphorothioate (PS) and 2'-O-methylated (2-OMe) PS oligonucleotides depends on the nature of the polymer additive in the CE background electrolyte. The selectivity of separation for different polymers increases in the line: linear polyacrylamide < polyethylene glycol < polyvinyl pyrrolidone. The separation of oligomer diastereomers was shown to be primarily based on the hydrophobic interaction with the polymer network that acts as a pseudostationary phase. While lowering the temperature resulted in improved separation, the addition of organic modifiers such as formamide, methanol or acetonitrile counteracts the solute adsorption on the polymer network, and decreases the selectivity of DNA diastereoseparation. The effect of molecular mass and concentration of the polymer on the separation selectivity was investigated.     

Analysis and applications of nanoparticles in the separation sciences: A case of gold nanoparticles

Nanometer-sized gold particles—gold nanoparticles (Au NPs)—are attracting a great deal of attention for their use in various technologies, including catalysis, optical and electronic devices, and separation science. In the emerging field of nanomaterials, the design, synthesis, and characterization of nanostructures are critical features because the manipulation of these structures has a direct effect on their resulting macroscopic properties. Nanostructures fabricated in layers on surfaces—for example, through self-assembly processes—have several potential applications in separation science. This review provides an introduction to the characterizations of Au NPs using size exclusion chromatography, high performance liquid chromatography (HPLC), and electrophoresis, and their self-assembly onto solid supports for analyses based on HPLC, gas chromatography, and capillary electrophoresis. In addition, sample concentration strategies involving the use of self-assembly approaches for surface modification of Au NPs are also discussed.     

Field amplified sample stacking coupled with chip-based capillary electrophoresis using negative pressure sample injection technique

A multi-T microchip for integrated field amplified sample stacking (FASS) with CE separation to increase the chip-based capillary electrophoresis (chip-based CE) sensitivity was developed. Volumetrically defined large sample plug was formed in one step within 5s by the negative pressure in headspace of the two sealed sample waste reservoirs produced using a syringe pump equipped with a 3-way valve. Stacking and separation can proceed only by switching the 3-way valve to release the vacuum in headspace of the two sample waste reservoirs. This approach considerably simplified the operations and the equipments for FASS in chip-based CE systems. Migration time precisions of 3.3% and 1.3% RSD for rhodamine123 (Rh123) and fluorescien sodium salt (Flu) in the separation of a mixture of Flu and Rh123 were obtained for nine consecutive determinations with peak height precisions of 4.8% and 3.4% RSD, respectively. Compared with the chip-based CE on the cross microchip, the sensitivity for analysis of FlTC, FITC-labeled valine (Val) and Alanine (Ala) increased 55-, 41- and 43-fold, respectively.     

Rapid prototyping of microfluidic devices with a wax printer

We demonstrate a rapid and inexpensive approach for the fabrication of high resolution poly(dimethylsiloxane) (PDMS)-based microfluidic devices. The complete process of fabrication could be performed in several hours (or less) without any specialized equipment other than a consumer-grade wax printer. The channels produced by this method are of high enough quality that we are able to demonstrate the sizing and separation of DNA fragments using capillary electrophoresis (CE) with no apparent loss of resolution over that found with glass chips fabricated by conventional photolithographic methods. We believe that this method will greatly improve the accessibility of rapid prototyping methods.     

Small molecule self‐assembly in polymer matrices

Using small molecules in polymer matrices is common in applications such as (i) plasticizing polymers to modify the glass transition and mechanical properties and (ii) dispersion of photoactive or electroactive small molecules in polymer matrices in organic‐electronic devices Aggregation of these small molecules and phase separation leading to crystallization often cannot be morphologically controlled. If these are designed with self‐assembling codes such as hydrogen bonding or aromatic interactions, their phase separation behavior would be distinctly different. This review summarizes the studies on morphologies in such situations, such as (i) sub‐surface assembly in polymer matrices, (ii) controlled polymerization‐induced phase separation to create polymer blends, (iii) using the polymer to direct the assembly of small molecules in liquid crystalline devices, (iv) functionalizing a polymer with self‐assembling small molecules to cause organo‐gelation which the polymer itself would not by itself, and (v) using such systems as templates to create porous polymer structures. Organic–inorganic hybrids using polymers as templates for nanostructures and imprinted porous membranes is an emerging area. Since self‐assembly is one of the dominating area of research with respect to both small molecules, polymers as well as the combination of the two, this review summarizes the studies on the aforementioned topics. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 451–478     

Nonconventional synthesis and characterization of ultrahigh-molar-mass polyacrylamides

In spite of the significant progresses in the field of replaceable sieving matrices for separating DNA in capillary electrophoresis (CE), an intense research activity is still going on to improve the separation of large size DNA sequencing fragments. There are evidences, both from experimental and theoretical sides that the resolution of these fragments, at the single base, requires the use of sieving matrices comprised of long chain linear polymers. In the separation of DNA fragments by CE are of upmost importance: (i) the complete solubility of the polymer, (ii) the linearity of the chain, (iii) the achievement of ultrahigh viscosity in dilute solutions. The aim of this work is the synthesis of ultrahigh-molecular-weight polymers which possess the three requirements mentioned above by employing a nonconventional method. We demonstrate that the sieving performance of polyacrylamide is directly correlated to its intrinsic viscosity.