Recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices

Cells are the fundamental unit of life, and studies on cell contribute to reveal the mystery of life. However, since variability exists between individual cells even in the same kind of cells, increased emphasis has been put on the analysis of individual cells for getting better understanding on the organism functions. During the past two decades, various techniques have been developed for single-cell analysis. Capillary electrophoresis is an excellent technique for identifying and quantifying the contents of single cells. The microfluidic devices afford a versatile platform for single-cell analysis owing to their unique characteristics. This article provides a review on recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices; focus areas to be covered include sampling techniques, detection methods and main applications in capillary electrophoresis, and cell culture, cell manipulation, chemical cytometry and cellular physiology on microfluidic devices.     

Microfluidic immunoaffinity separations for bioanalysis

Microfluidic devices often rely on antibody-antigen interactions as a means of separating analytes of interest from sample matrices. Immunoassays and immunoaffinity separations performed in miniaturized formats offer selective target isolation with minimal reagent consumption and reduced analysis times. The introduction of biological fluids and other complicated matrices often requires sample pretreatment or system modifications for compatibility with small-scale devices. Miniaturization of external equipment facilitates the potential for portable use such as in patient point-of-care settings. Microfluidic immunoaffinity systems including capillary and chip platforms have been assembled from basic instrument components for fluid control, sample introduction, and detection. The current review focuses on the use of immunoaffinity separations in microfluidic devices with an emphasis on pump-based flow and biological sample analysis.     

Highly efficient protein separations in high-performance capillary electrophoresis,using hydrophilic coatings on polysiloxane-bonded columns

Three methods for preparing hydrophilic coatings on polysiloxane bonded CElect H-type capillary electrophoresis columns have been shown. The polyalkylsiloxane-bonded phase is the first coating layer on the capillary surface, and nonionic surfactant, hydrophilic polymer, or polymer surfactant, adsorbed onto this first layer through hydrophobic interactions, forms the second coating layer. The resultant capillary surfaces are inert, hydrophilic, and suitable for highly efficient protein separations. The effectiveness and applicability of these capillary surface modification methods were tested for the separations of a variety of proteins over a wide range of buffer pH values under different capillary electrophoretic operation modes.     

Fluorescence correlation spectroscopy for ultrasensitive DNA analysis in continuous flow capillary electrophoresis

Continuous flow capillary electrophoresis (CFCE) is non-separations based analytical technique based on the free solution electrophoretic mobility of biological molecules such as DNA, RNA, peptides, and proteins. The electrophoretic mobilities and translational diffusion constants of the analyte molecules are determined using single molecule detection methods, including fluorescence correlation spectroscopy (FCS). CFCE is used to resolve multiple components in a mixture of analytes, measure electrophoretic mobility shifts due to binding interactions, and study the hydrodynamic and electrostatic properties of biological molecules in solution. Often this information is obtained with greater speed and sensitivity than conventational separations-based capillary-zone electrophoresis. This paper will focus on the application of two-beam fluorescence cross-correlation spectroscopy as a versatile detection method for CFCE and explore several applications to the study of the solution properties of single-stranded DNA.     

High-speed microchip electrophoresis method for the separation of (R,S)-naproxen

In this research, a capillary electrophoretic method for the fast enantiomeric resolution of (R,S)-naproxen was investigated. Method development involved variation of applied potential, buffer concentration, buffer pH, and cyclodextrin concentration. The optimum electrophoretic separation conditions were 110 mM sodium acetate run buffer (pH 6.0), 30 mM methyl-beta-cyclodextrin, 20% (v/v) acetonitrile, 25 degrees C. The total length of capillary was 48 cm, (50 microm I.D.) with ultra violet (UV) detection at 232 nm. Using these conditions, the number of theoretical plates was close to one million (896,000/m). The possibility of achieving a fast chiral separation of (R,S)-naproxen on a microchip of 2.5 cm in length was investigated. Complete enantiomeric resolution of naproxen was achieved in less than 1 min, on this microchip platform, with linear imaging UV detection. This system had the advantage of real-time separation monitoring, so that enantiomeric resolution could be visually observed, and high-speed chiral analysis was realized. The microchip electrophoresis (MCE) separation was compared with the capillary electrophoresis (CE) separation with regards to speed, efficiency, separation platform, and precision. This work highlights the potential of CE and MCE in future chiral separations.     

Noncovalent labeling of proteins in capillary electrophoresis with laser-induced fluorescence detection

Interest in the use of capillary electrophoresis (CE) as a tool for protein separations continues to grow. Additionally, laser-induced fluorescence (LIF) detection schemes promise ultrasensitive detection of small quantities of these important biomolecules following their separation. In most cases, LIF detection of proteins necessitates their prior derivatization with a fluorescent label molecule. To minimize the amount of additional sample handling and time associated with such labeling procedures, not to mention the sometimes-stringent pH and temperature controls they require, noncovalent labeling is presented as a viable alternative. This review article considers established methods for noncovalent labeling of proteins for their subsequent analysis by CE-LIF. Label molecules suitable for excitation and emission in the ultraviolet, visible, and near-infrared regions of the spectrum are enumerated for a variety of protein analytes.     

Application of microfluidic chip with integrated optics for electrophoretic separations of proteins

This paper describes the fabrication, the characterization and the applications of a capillary electrophoresis microchip. This hybrid device (glass/PDMS) features channels and optical waveguides integrated in one common substrate. It can be used for electrophoretic separation and fluorimetric detection of molecules. The microfluidic performance of the device is demonstrated by capillary zone and gel electrophoresis of proteins.     

Cancer Liquid Biopsy Using Integrated Microfluidic Exosome Analysis Platforms

Liquid biopsies serve as both powerful noninvasive diagnostic tools for early cancer screening and prognostic tools for monitoring cancer progression and treatment efficacy. Exosomes are promising biomarkers for liquid biopsies, since these nano‐sized extracellular vesicles (EVs) enrich proteins, lipids, mRNAs, and miRNAs from cells of origin, including cancer cells. Although exosomes are abundantly present in various bodily fluids, conventional exosome isolation and detection methods that rely on benchtop equipment are time‐consuming, expensive, and involve complicated non‐portable procedures. As an alternative, recently developed microfluidic platforms can perform effective exosome separation and detection for liquid biopsies using a single device. Such methods offer advantages of integrity, speed, cost‐efficiency, and portability over conventional benchtop and early microfluidic‐based single‐functional methods which can only separate or detect exosomes separately. These advances have made exosome‐based point‐of‐care (POC) applications possible. This review outlines recent integrated microfluidic‐based exosomal detection strategies to guide future development of such devices for use in liquid biopsies for early cancer screening, prognostic monitoring, and other potential POC applications.     

Development of a full‐length human protein production pipeline

There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high‐throughput (HT) methods, we transferred the genes of 31 full‐length proteins into three expression vectors, and expressed the collection as N‐terminal HaloTag fusion proteins in Escherichia coli and two commercial cell‐free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip^® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full‐length human proteins in these three expression systems.     

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.     

A Method for the Electrophoretic Analysis of Proteins and RNAs of Single Cells*

SYNOPSIS A method is described for the electrophoretic analysis of proteins or RNAs from individual amebae. The method is based on fluorographic autoradiography of semi-micro polyacrylamide gels in which [³⁵S]methionine or [³H]uridine materials from single cells have been subjected to electrophoresis. The method is more sensitive and provides better resolution than previous methods for single cells. It is suitable, also, for quantitation of the separated components.     

Simple luminescence detectors using a light-emitting diode or a Xe lamp, optical fiber and charge-coupled device, or photomultiplier for determining proteins in capillary electrophoresis: a critical comparison

The performance of two homemade fluorescence-induced capillary electrophoresis detectors, one based on light-emitting diode (LED) as the excitation source and a charge-coupled device (CCD) photodetector and the other based on a commercial luminescence spectrometer (Xe lamp) as the excitation source and a photomultiplier tube as a detector, were compared for the determination of fluorescent proteins R-phycoerythrin and B-phycoerythrin. Both devices use commercially available, reasonably priced optical components that can be used by nonexperts. After fine optimization of several optical and separation parameters in both devices, a zone capillary electrophoresis methodology was achieved with 50mM borate buffer (pH 8.4) and 10mM phytic acid for the determination of two phycobiliproteins. Detection limits of 0.50 and 0.64microg/ml for R-phycoerythrin and B-phycoerythrin, respectively, were achieved by using the LED-induced fluorescence capillary electrophoresis (LED-IF-CE) system, and corresponding detection limits of 2.73 and 2.16microg/ml were achieved by using the Xe lamp-IF-CE system. Analytical performance and other parameters, such as cost and potential to miniaturization, are compared for both devices.     

基于电感耦合等离子体质谱的单细胞分析

单细胞分析可以获得细胞在微环境中准确的个体信息,对于研究细胞的信号传导、生理病理和疾病的早期诊断等具有十分重要的意义.近年来,基于电感耦合等离子体质谱(ICP-MS)的单细胞分析方法开始得到越来越多的应用.本文综述了基于ICP-MS的单细胞分析方法及其在免疫分析、疾病诊断、药物筛选、纳米分析等方面的部分应用,并对基于ICP-MS的单细胞分析方法做出总结和展望.     

A capillary electrophoretic method for monitoring the presence of alpha-tubulin in nuclear preparations

A sensitive capillary electrophoretic method was developed to detect the presence of alpha-tubulin, a microtubular cytoskeletal component, in isolated nuclear preparations. These preparations are treated with anti-alpha-tubulin primary mouse antibodies and then stained with a fluorescently labeled anti-mouse IgG antibody. The stained preparation is then analyzed by capillary electrophoresis with laser-induced fluorescence detection, a technique that allows for sensitive detection of fluorescently labeled species. Using this method, it is feasible to count individual subcellular aggregates containing alpha-tubulin (SATs), estimate the number of alpha-tubulin molecules per SAT, determine the cumulative intensity of all SATs as an estimate of the relative level of alpha-tubulin in a preparation, and obtain their apparent electrophoretic mobility distribution. The method was validated by comparing SATs from untreated cells with those from colchicine-treated cells. Since colchicine is a microtubule-disrupting agent, treatment reduced the number of SATs per cell as well as the cumulative intensity of all SATs in a preparation. In contrast, the apparent electrophoretic mobility distribution was not influenced by colchicine treatment, suggesting that this parameter is not strongly dependent on the alpha-tubulin content. Given the zeptomolar sensitivity of laser-induced fluorescence detection and the widespread availability of antibodies, the approach used here represents an improvement in the detection of cytoskeletal impurities in subcellular fractions.     

Studies of reversible inhibition,irreversible inhibition,and activation of alkaline phosphatase by capillary electrophoresis

Reversible inhibition, irreversible inhibition, and activation of calf intestinal alkaline phosphatase (EC 3.1.3.1) have been studied by capillary electrophoresis. The capillary electrophoretic enzyme-inhibitor assays were based on electrophoretic mixing of inhibitor and enzyme zones in a substrate-filled capillary. Enzyme inhibition was indicated by a decrease in product formation detected in the capillary by laser-induced fluorescence. Reversible enzyme inhibitors could be quantified by Michaelis-Menten treatment of the electrophoretic data. Reversible, competitive inhibition of alkaline phosphatase by sodium vanadate and sodium arsenate has been examined, and reversible, noncompetitive inhibition by theophylline has been studied. The K(i) values determined for these reversible inhibitors using capillary electrophoresis are within the range of values reported in the literature for the same enzyme-inhibitor combinations. Irreversible inhibition of alkaline phosphatase by EDTA at concentrations of 1.0mM and above has been observed. Activation of alkaline phosphatase has also been observed for EDTA at concentrations from 20 to 400 microM.     

微流控器件-质谱联用接口技术研究进展与应用

生物分析是生命科学研究中的重要环节,分析仪器的小型化是提高生物分析灵敏度、速度、通量和降低成本的有效途径之一.微流控技术能够方便地操纵微量样品,具有集成度高、样品耗量小、污染少等诸多其他常量流控技术难以具备的优点,适用于进行多通道样品处理和高通量分析.除广泛采用的光学和电化学检测手段外,质谱也被用作这些微流控器件的检测器,并逐渐形成了微流控器件-质谱联用技术专门研究领域,进一步促进了自动化程度好、灵敏度高、特异性强的高通量生物分析方法的迅速发展.在大量调研国内外文献的基础上,对微流控器件-质谱联用领域的研究背景和现状进行了综述,不但介绍了微流控器件的制造技术还着重介绍了微流控器件-质谱联用技术在蛋白质组学等生物质谱分析方面的应用和新近进展,评述了可能的发展趋势.     

Profile--Richard A. Mathies. Interview by Suzanne Berry.

Richard A. Mathies (Fig. 1) is a professor of chemistry at the University of California (UC) at Berkeley. His early work at UC was on the use of resonance Raman and time-resolved optical spectroscopy to elucidate the structure and reaction dynamics of energy and information-transducing photoactive proteins called rhodopsins. His work on the Human Genome Project led to the development of high-throughput platform technologies including capillary array electrophoresis and energy transfer fluorescent dye labels for DNA sequencing and analysis. He has also pioneered the development of microfabricated capillary electrophoresis devices, capillary array electrophoresis microplates and microfabriated integrated sample preparation and detection methods. He is the co-founder of the Center for Analytical Biotechnology at UC Berkeley. Mathies was interviewed at the BIOMEMS and Biomedical Nanotechnology conference in Columbus, Ohio, 21-25 September 2001, where he gave a talk about capillary array electrophoresis-based microprocessors. Such devices could be used as point-of-care clinical and genetic analyzers, in integrated microfluidic sequencing chips and in DNA-based computing.     

Ultrasensitive protein-DNA binding assays

Protein-DNA binding assays have been used in a variety of fields from fundamental studies regarding the binding process itself, to serving as probes for the detection, quantification and separation of target analytes. These assays have been used for the study of protein-DNA complex stoichiometry, the detection of DNA damage, and real-time separation of free and bound complexes by electrophoretic mobility. Synthetic DNA oligonucleotides, known as aptamers, have been increasingly used for affinity binding assays to proteins, as well as for separation studies and as biosensors. Recent advances have been made in protein-DNA binding assays using capillary electrophoresis, laser-induced fluorescence, fluorescence polarization, molecular beacons, and affinity chromatography.     

Enantioselective separations using capillary electrophoresis

The preconditions are outlined for enantioselective separations in capillary electrophoresis (CE) with chiral selectors as additives to the background electrolyte. Free solution capillary electrophoresis conditions are characterised by a single solution phase. Chiral separations are reviewed by selector type (chiral ligand exchange, cyclodextrins, crown ethers, glycoproteins) with the extensive studies on cyclodextrins grouped into sections on amino acids, pharmaceuticals, and speciality chemicals, optimisation, biological fluids, and quantitative aspects. In micellar electrokinetic capillary chromatography, enantioselective discrimination occurs by partition in a two-phase system, with a chiral micellar phase as selector. Optimum separation conditions can be readily predicted for a given selector–selectand combination, and absolute values of binding constants determined by CE. Advantages of CE in comparison with HPLC using a chiral stationary phase include robust, rapid assays and the use of small volumes of aqueous solutions; disadvantages include less favourable detection limits. © 1994 Wiley-Liss, Inc.     

Analysis of intracellular reducing levels in human hepatocytes on three‐dimensional focusing microchip

A novel three‐dimensional hydrodynamic focusing microfluidic device integrated with high‐throughput cell sampling and detection of intracellular contents is presented. It has a pivotal role in maintaining the reducing environment in cells. Intracellular reducing species such as vitamin C and glutathione in normal and tumor cells were labeled by a newly synthesized 2,2,6,6‐tetramethyl‐piperidine‐1‐oxyl‐based fluorescent probe. Hepatocytes are adherent cells, which are prone to attaching to the channel surface. To avoid the attachment of cells on the channel surface, a single channel microchip with three sheath‐flow channels located on both sides of and below the sampling channel was developed. Hydrostatic pressure generated by emptying the sample waste reservoir was used as driving force of fluid on the microchip. Owing to the difference between the liquid levels of the reservoirs, the labeled cells were three‐dimensional hydrodynamically focused and transported from the sample reservoir to the sample waste reservoir. Hydrostatic pressure takes advantage of its ease of generation on a microfluidic chip without any external pressure pump, which drives three sheath‐flow streams to constrain a sample flow stream into a narrow stream to avoid blockage of the sampling channel by adhered cells. The intracellular reducing levels of HepG2 cells and L02 cells were detected by home‐built laser‐induced fluorescence detector. The analysis throughput achieved in this microfluidic system was about 59–68 cells/min. Copyright © 2013 John Wiley & Sons, Ltd.