Quantitative determination of dopamine in single rat pheochromocytoma cells by microchip electrophoresis with only one high-voltage power supply.

We developed a method for the direct identification of dopamine in single cultured rat pheochromocytoma cells by capillary electrophoresis using an end-channel carbon fiber nanoelectrode amperometric detector. The operation mode was designed to achieve single-cell injection and lysis in microfluidic chip electrophoresis with only one high-voltage power supply. The separation and detection conditions were optimized. Four catecholamines were baseline-separated and determined with this system, and the cell density and liquid height of the reservoirs were accommodated for single cell loading, docking and analysis. The microchip capillary electrophoresis system was successfully applied to determine dopamine in single cultured rat pheochromocytoma cells.

Integrated microdevice for long-term automated perfusion culture without shear stress and real-time electrochemical monitoring of cells.

Electrochemical techniques based on ultramicroelectrodes (UMEs) play a significant role in real-time monitoring of chemical messengers' release from single cells. Conversely, precise monitoring of cells in vitro strongly depends on the adequate construction of cellular physiological microenvironment. In this paper, we developed a multilayer microdevice which integrated high aspect ratio poly(dimethylsiloxane) (PDMS) microfluidic device for long-term automated perfusion culture of cells without shear stress and an independently addressable microelectrodes array (IAMEA) for electrochemical monitoring of the cultured cells in real time. Novel design using high aspect ratio between circular "moat" and ring-shaped micropillar array surrounding cell culture chamber combined with automated "circular-centre" and "bottom-up" perfusion model successfully provided continuous fresh medium and a stable and uniform microenvironment for cells. Two weeks automated culture of human umbilical endothelial cell line (ECV304) and neuronal differentiation of rat pheochromocytoma (PC12) cells have been realized using this device. Furthermore, the quantal release of dopamine from individual PC12 cells during their culture or propagation process was amperometrically monitored in real time. The multifunctional microdevice developed in this paper integrated cellular microenvironment construction and real-time monitoring of cells during their physiological process, and would possibly provide a versatile platform for cell-based biomedical analysis.

[Microchip electrochromatography: the latest developments and applications].

This review summarizes recent developments and applications of microchip electrochromatography (microCEC) mainly in the past five years between 2005 and 2009 with a focus on column technologies. In addition, some new improvements in the chip design and fabrication, sample preconcentration, electroosmotic flow control as well as mechanisms that govern electrochromatographic separation are described and reviewed. The features and limitations of several practical aspects of their applications are highlighted.

Development and evaluation of a rotary cell for capillary electrophoresis-chemiluminescence detection.

Many efforts have been made toward the advancement of capillary electrophoresis chemiluminescence (CE-CL) detection and its applications through continuous development and improvement of interfaces. In this study, a novel rotary cell for CE-CL detection was fabricated and evaluated. A ring-shaped narrow channel with a quartz bottom is made in a cell body to hold CL reactants and act as the reaction chamber. The CE capillary is placed closely to the bottom of the reaction chamber where analyte is deposited into the CL reactants for reactions to occur. Detection is achieved with a photomultiplier tube below the reaction channel. An advantage of the rotary reaction cell is that it renews the reactants at the capillary end as it revolves at a preset speed during experiments. The rotary detection cell presents a new concept to the conventional coaxial-flow configuration by solving the problems of bubble formation and flow blockage that often interrupt the electrophoresis. Two standard proteins, horseradish peroxidase (HRP) and hemoglobin (Hb), were used to evaluate the interface's performance with luminol/H(2)O(2) CL system. Satisfactory sensitivities (LOD of 0.91 x 10(-9) M for HRP, and 4.37 x 10(-8) M for Hb at S/N = 3) were obtained in this proof-of-concept experiment. This novel design provides a straightforward and robust interface for CE-CL hyphenation.

Real-time monitoring of oxidative burst from single plant protoplasts using microelectrochemical sensors modified by platinum nanoparticles.

Oxidative bursts from plants play significant roles in plant disease defense and signal transduction; however, it has not hitherto been investigated on individual living plant cells. In this article, we fabricated a novel sensitive electrochemical sensor based on electrochemical deposition of Pt nanoparticles on the surface of carbon fiber microdisk electrodes via a nanopores containing polymer matrix, Nafion. The numerous hydrophilic nanochannels in the Nafion clusters coated on the electrode surface served as the molecular template for the deposition and dispersion of Pt, which resulted in the uniform construction of small Pt nanoparticles. The novel sensor displayed a high sensitivity for detection of H(2)O(2) with a detection limit of 5.0 x 10(-9) M. With the use of this microelectrochemical sensor, the oxidative burst from individual living plant protoplasts have been real-time monitored for the first time. The results showed that oxidative burst from single protoplasts triggered by a pathogen analogue were characterized by quanta release with a large number of "transient oxidative microburst" events, and protoplasts from the transgenic plants biologically displayed better disease-resistance and showed a distinguished elevation and longer-lasting oxidative burst.

Monitoring of vesicular exocytosis from single cells using micrometer and nanometer-sized electrochemical sensors.

Communication between cells by release of specific chemical messengers via exocytosis plays crucial roles in biological process. Electrochemical detection based on ultramicroelectrodes (UMEs) has become one of the most powerful techniques in real-time monitoring of an extremely small number of released molecules during very short time scales, owing to its intrinsic advantages such as fast response, excellent sensitivity, and high spatiotemporal resolution. Great successes have been achieved in the use of UME methods to obtain quantitative and kinetic information about released chemical messengers and to reveal the molecular mechanism in vesicular exocytosis. In this paper, we review recent developments in monitoring exocytosis by use of UMEs-electrochemical-based techniques including electrochemical detection using micrometer and nanometer-sized sensors, scanning electrochemical microscopy (SECM), and UMEs implemented in lab-on-a-chip (LOC) microsystems. These advances are of great significance in obtaining a better understanding of vesicular exocytosis and chemical communications between cells, and will facilitate developments in many fields, including analytical chemistry, biological science, and medicine. Furthermore, future developments in electrochemical probing of exocytosis are also proposed.

Real-time monitoring of NO release from single cells using carbon fiber microdisk electrodes modified with single-walled carbon nanotubes.

In this paper, a novel NO electrochemical microsensor, which is fabricated by modifying the surface of a carbon fiber microdisk electrode (CFMDE, diameter: 5-7 microm) with single-walled carbon nanotubes (SWNTs) and Nafion membrane, is reported for the first time. The modification of SWNTs dramatically improves the sensitivity of CFMDEs, and the detection limit for NO is 4.3 nM that is nearly 10 times lower than that from the bare one and lower than most NO electrochemical sensors reported before. The Nafion membrane offers a good barrier to some interferents such as nitrite and ascorbic acid without losing response speed to NO. The sensor has been successfully applied to the measurement of NO release from single isolated human umbilical vein endothelial cells (HUVECs). Real-time amperometric data show that the addition of l-arginine (l-arg) or acetylcholine (ACh) can cause a quick increase in NO production with a maximum concentration of 232+/-44 nM (n=5) and 159+/-29 nM (n=5), respectively.

Analysis of amino acids in human vascular endothelial (ECV-304) cells by microchip electrophoresis with fluorescence detection.

A rapid and sensitive method was developed for the analysis of amino acids by microchip electrophoresis with Hg-lamp excitation fluorescence detection. Fluorescein-isothiocyanate (FITC) was chosen to estimate the sensitivity of this system, and the detection limit (S/N = 3) with FITC was 1.7 nM, which showed that the system was sensitive as well as simple. Two derivatizing agents, FITC and ortho-phthalaldehyde (OPA) were employed to label amino acids and were compared in the same fluorescence detection system with an Hg lamp as the excitation source. The separation parameters were optimized in detail. Optimum separation of OPA-labeled amino acids was obtained in less than 200 s with 20 mM borate buffer (pH 9.0) containing 20% acetonitrile and 10 mM beta-cyclodextrin. Detection limits for amino acids (alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp)) of 0.38-1.0 muM were achieved. The method was successfully applied to analysis of amino acids in human vascular endothelial cells (ECV-304). The average amount of amino acids in single ECV-304 cells is estimated to be 5.84 fmol for Ala, 2.78 fmol for Tau, 1.15 fmol for Gly, 3.10 fmol for Glu, and 1.30 fmol for Asp.

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.

Analysis of inorganic and small organic ions by CE with amperometric detection.

Capillary electrophoresis has become a widely useful analytical technology. Amperometric detection is extensively employed in capillary electrophoresis for its many inherent virtues, such as rapid response, remarkable sensitivity, and low cost of both detectors and instrumentations. Analysis of inorganic and small organic ions by capillary electrophoresis is an important research field. This review focuses on the recent developments of capillary electrophoresis coupled with amperometric detection for analysis of inorganic and small organic ions. Advancements in electrophoresis separation modes, amperometric detection modes, working electrodes, and applications of inorganic ions, amino acids, phenols, and amines are discussed.

Indirect determination of pyruvic acid by capillary electrophoresis with amperometric detection.

A method of indirectly measuring pyruvic acid (PA) by capillary electrophoresis with amperometric detection is proposed for the first time. It is based on the oximation reaction between PA and hydroxylamine (NH(2)OH), and the quantification of PA was performed by direct and sensitive amperometric detection of excessive NH(2)OH after the oximation reaction. This method displayed a good sensitivity, and the detection limits of NH(2)OH and PA are 1.76 x 10(-7) and 3.88 x 10(-7)mol/L, respectively at S/N=3. The linear relationship between the peak current and PA concentration is exhibited over the range from 4 x 10(-6) to 1 x 10(-4)mol/L. This method has been applied to determine PA in rat plasma with satisfactory results.

Imaging and detection of morphological changes of single cells before and after secretion using scanning electrochemical microscopy.

Images of Human umbilical vein endothelial cells (HUVECs) have been obtained and the regulation of cell morphology changes after nitric oxide release has been recorded and discerned quantitatively for the first time using scanning electrochemical microscopy.

Capillary electrophoretic immunoassay for alpha-fetoprotein with chemiluminescence detection.

A capillary electrophoretic immunoassay with chemiluminescence detection (CEIA-CL) using a non-competitive format for analyzing tumor marker alpha-fetoprotein (AFP) has been developed. In this method, antigen (Ag) AFP reacts with an excess amount of horseradish peroxidase (HRP)-labeled antibody (Ab*). The free Ab* and the bound Ab*-Ag complex produced in the solution are separated by CE in a separation capillary. Then they catalyze the reaction of enzyme substrate luminol and H(2)O(2) in a reaction capillary following the separation capillary. Parameters affecting the CE separation and CL detection were investigated. Under the optimal conditions, the free Ab* and the Ab*-Ag complex were well separated within 4 min, the linear range and the detection limit (S/N=3) for AFP were 5-500 ng/ml and 0.85 ng/ml (1.2 x 10(-11)M), respectively. The proposed method has been applied satisfactorily in the analysis of human sera samples.

[Studies on single-cell analysis].

The single-cell analysis is a frontier field of sciences that has been developed from the interdisciplinary collaborations of analytical chemistry, biology and medicine. At the present time, the great achievements in the application of capillary electrophoresis and microfluidic chip to single-cell analysis, particularly, in the application of microfluidic chip to culture, manipulation, location, separation and detection of components of single cells, real-time detection of secretion from single-cells and high-throughput array detection have been made. The unit procedures can be made up in any device in accordance with the requirements. In this paper, the work of the author's group is presented mainly. In addition, the developments of the capillary electrophoresis and chip electrophoresis applied to single-cell analysis in recent years are reviewed. The prospects for single-cell analysis including capillary electrophoresis and microfluidic chip, measurement and control at the cell-chip interface, and quantum dots for probing live cells are also proposed.

Carbon fiber nanoelectrodes applied to microchip electrophoresis amperometric detection of neurotransmitter dopamine in rat pheochromocytoma (PC12) cells.

Microelectrodes have been adopted in electrochemical detection for CE or microchip CE in recent years. In this paper, the use of nanoelectrodes (with tip diameter of 100-300 nm) as the electrochemical detector in microchip CE is firstly reported. The experimental results indicated that both the sensitivity and resolution of microchip CE with the carbon fiber nanoelectrode (CFNE) amperometric detection have been improved markedly comparing with the traditional microelectrodes. The detection limit of dopamine (S/N = 3) is 5.9x10(-8) M, which is one or two orders of magnitude lower than that reported so far, and the resolution of dopamine (DA) and isoprenaline (IP) has also improved from 0.6 (using 7 mum carbon fiber microelectrodes, CFME) to 1.0. We assembled a novel and easily operated microchip CE system with end-column amperometric detection, which allows the convenient and fast replacement of the passivated electrodes. Under the optimized condition, the RSDs of peak height and migration time are 1.47 and 0.31%, respectively (n = 40), indicating that the system displays excellent reproducibility. The nanoelectrode-based microchip CE system has been successfully applied to the determination of DA in cultured rat pheochromocytoma (PC12) cells, and the average content of DA in an individual PC12 cell is 0.54 +/- 0.07 fmol, which is in good agreement with that reported in the literature.

Determination of neurotransmitters in PC 12 cells by microchip electrophoresis with fluorescence detection.

A sensitive fluorescence detection system with an Hg-lamp as the excitation source and a photon counter as the detector for microchip CE (MCE) has been developed. O-Phthaldialdehyde (OPA, lambda(ex) = 340 nm) was employed to label the catecholamine neurotransmitters such as dopamine (DA), norepinephrine (NE), and amino acid neurotransmitters including alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp). The separation of seven derivatized neurotransmitters was successfully performed in MCE and the detection limits (S/N = 3) for DA, NE, Ala, Tau, Gly, Glu, and Asp were 0.85, 0.49, 0.23, 0.15, 0.13, 0.18, and 0.29 fmol, respectively. The system was then successfully applied for separation and determination of neurotransmitters in rat pheochromocytoma (PC 12) cells, and the average amounts of analyte per cell from a cell population were 2.5 fmol for DA, 3.3 fmol for Ala, 8.2 fmol for Tau, 4.0 fmol for Gly, and 1.9 fmol for Glu, respectively. By single-cell injection mode, electrophoresis separation and quantitative measurement of Glu in individual PC 12 cells was obtained. The average value of Glu per cell from single PC 12 cells analysis was found to be 3.5 +/- 3.1 fmol.

Monitoring the reaction of hemoglobin with hydrogen peroxide by capillary electrophoresis-chemiluminescence detection.

The reaction of hemoglobin (Hb) with hydrogen peroxide (H2O2) leads to fluorescent product and heme degradation. We applied capillary electrophoresis-chemiluminescence (CE-CL) detection to monitor the course of Hb reacting with H2O2. Hb and released free iron ion (Fe3+) were detected based on their enhancement effects on CL of the luminol-H2O2 system. In this study, we discovered an intermediate of this reaction which intensely enhances the luminol-H2O2 CL system. The ratio of max CL signals of Fe3+, Hb and this intermediate is circa 1:10:60.

CE-based analysis of hemoglobin and its applications in clinical analysis.

This review focuses on the developments and trends in CE including CIEF, CZE, MEKC, two-dimensional conjunction of CIEF-capillary gel electrophoresis, and MEKC-CZE on microfluidic devices coupled to different detection approaches, such as UV absorbance, LIF, MS, and chemiluminescence etc. for performing analysis of hemoglobin (Hb), also with an emphasis on its applications in clinical analysis. Analysis of human Hb is of important clinical sense for numerous hemoglobinopathies associated with the congenital defects and abnormal contents of Hb. The diversiform modes render CE a comprehensive primary clinical tool for Hb analysis, which is rapid, sensitive, high-resolution, and not labor-intensive.

Monitoring dopamine release from single living vesicles with nanoelectrodes.

Carbon fiber nanoelectrodes (tip diameter = ca. 100 nm) have been first used to monitor real-time dopamine release from single living vesicles of single rat pheochromocytoma (PC12) cells. The experiments show that active and inactive release sites exist on the surface of cells, and the spatial distributions have been differentiated even in the same active release zone. It is first demonstrated that multiple vesicles can sequentially release dopamine at the same site of the cell surface, which possibly plays the main role in the dopamine release from PC12 cells.

Capillary electrophoresis immunoassay chemiluminescence detection of zeptomoles of bone morphogenic protein-2 in rat vascular smooth muscle cells.

A capillary electrophoresis immunoassay (CEIA) method based on enhanced chemiluminescence (CL) detection was developed and applied to arteriosclerosis pathology research in the medical field. The system of enzyme-horseradish peroxidase (HRP) catalyzing the luminol/H2O2/p-iodophenol reaction was adopted in this paper. HRP was detected with the detection limit (S/N = 3) of 4.4 pM (53 zmol), which represents one of the highest sensitivities of HRP reported yet. HRP was first linked to bone morphogenic protein-2 (BMP-2) in rat vascular smooth muscle (VSM) cells with noncompetitive format and analyzed by CE-CL. HRP-Ab(2)-mAb-BMP-2 complexes can be baseline separated from free HRP in 3 min. The detection limit (S/N = 3) of BMP-2 is 6.2 pM (75 zmol). This technique has been successfully applied to arteriosclerosis development mechanistic study by investigating the change of BMP-2 content in VSM cells, which were stimulated by angiotensin II for different times. The change trends of BMP-2 contents are well in accord with that of the commonly used pathology image analysis system. It proves that the CEIA-CL technique proposed could be developed into a sensitive and new method for clinical assay and pathology research.