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.

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.

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.

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.

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.

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.

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.

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.

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.

Transport, location, and quantal release monitoring of single cells on a microfluidic device.

A novel microfluidic device has been developed for on-chip transport, location, and quantal release monitoring of single cells. The microfluidic device consists of a plate of PDMS containing channels for introducing cells and stimulants and a glass substrate into which a cell micro-chamber was etched. The two tightly reversibly sealed plates can be separated for respective cleaning, which significantly extends the lifetime of the microchip that is frequently clogged in cell analysis experiments. Using hydraulic pressure, single cells were transported and located on the microfluidic chip. After location of a single PC12 cell on the microfluidic chip, the cell was stimulated by nicotine that was also introduced through the micro-channels, and the quantum release of dopamine from the cell was amperometricly detected with our designed carbon fiber microelectrode. The results have demonstrated the convenience and efficiency of using the microfluidic chip for monitoring of quantal release from single cells and have offered a facile method for the analysis of single cells on microfluidic devices.

Carbon fiber nanoelectrodes modified by single-walled carbon nanotubes.

Microelectrode voltammetry has been considered to be a powerful technique for single biological cell analysis and brain research. In this paper, we have developed a simple method to get highly sensitive carbon fiber nanoelectrodes (CFNE) modified by single-walled carbon nanotubes (SWNTs) on the basis of our previous work. The electrochemical behavior of SWNTs/CFNE was characterized by potassium ferricyanide, dopamine (DA), epinephrine (E), and norepinephrine (NE) using cyclic voltammetry (CV). Compared with CFNE, SWNTs/CFNE has a much larger available internal surface area per external geometric area, which is supported by SEM images. The modified electrodes show very high sensitivity and favorable electrochemical behavior toward these neurotransmitters. The peak current increases linearly with the concentration of DA, E, and NE in the range of 1.0 x 10(-)(7)-1.0 x 10(-)(4), 3.0 x 10(-)(7)-1.0 x 10(-)(4), and 5.0 x 10(-)(7)-1.0 x 10(-)(4) M, respectively. The CV detection limit (S/N = 3) of DA, E, and NE is 7.7 x 10(-)(9), 3.8 x 10(-)(8), and 4.2 x 10(-)(8) M, respectively. The modified electrode exhibited almost the same electrochemical behavior after 15 days, indicating that SWNTs/CFNE is pretty stable and has good reproducibility.

Separation of biogenic amines by micellar electrokinetic chromatography with on-line chemiluminescence detection.

A method of on-line chemiluminescence detection with capillary electrophoresis for biogenic amines (diaminopropane, putrescine, cadaverine and diaminohexane) labeled with N-(4-aminobutyl)-N-ethylisoluminol is reported for the first time. Two separation modes, capillary zone electrophoresis and micellar electrokinetic chromatography (MEKC), were studied. The results show that excellent resolution was achieved in MEKC. Parameters affecting separation process and chemiluminescence detection have been examined in detail. Under the optimum conditions, the baseline separation of four amines was obtained within 7.5 min. The detection limits (S/N=3) of diaminopropane, putrescine, cadaverine and diaminohexane are 3.5 x 10(-8), 3.5 x 10(-8), 3.9 x 10(-8) and 1.2 x 10(-7) M, respectively. The method was applied to the analysis of biogenic amines in lake water.

Elemental speciation analysis in capillary electrophoresis.

The growing awareness of the strong development of the toxicity of heavy metals upon their chemical forms has led to an increasing interest in the qualitative and quantitative determination of specific metal species. Speciation has therefore become an important topic of present-day analytical research. The development in the elemental speciation analysis by capillary electrophoresis (CE) is reviewed. Various CE separation modes and detection techniques applied are discussed. A comprehensive description of reported methods to date in CE speciation analysis including metals, metalloids and nonmetallic elements is demonstrated. Some examples are presented to demonstrate CE's ability to solve real-world speciation analysis with emphasis on the applications in biological and environmental samples. Further, some issues concerning the limitations and the future of CE with regard to speciation studies are also discussed.

Ultrasensitive chemiluminescence detection in capillary electrophoresis.

Capillary electrophoresis techniques offer high plate numbers and are highly suited for the efficient separations of a wide variety of chemical components in diverse matrices. Because of the small capillary and detection cell dimensions, together with the minute volumes of samples to be injected, sensitive detection schemes based on different physicochemical principles are being developed. One logical approach to increased sensitivity in capillary electrophoresis detection has been the development of chemiluminescence-based detectors. The development of on-line ultrasensitive chemiluminescence detection (referred to the concentration detection limit of nM order of magnitude or mass detection limit of amol order of magnitude) in capillary electrophoresis system is reviewed. The applications and limitations of the current detection methodology are briefly considered and future prospects for the development are discussed.

Influence of soluble polymer polyvinylpyrrolidone on separation of small peptides and amino acids by microchip-based capillary electrophoresis.

In microchip-based capillary electrophoresis, the resolution and separation efficiency of small peptides and amino acids can be noticeably improved by adding a low molecular weight (30,000) soluble polymer additive, polyvinypyrrolidone in the separation medium. Several separation conditions such as injection time and electrophoretic buffer have been investigated and optimized. Using an electro-stacking scheme, the resolution and separation efficiency of small peptides and amino acids can be enhanced significantly. Under the optimal conditions, the separation of fluorescein isothiocyanate Isomer I-labeled small peptides and amino acids was successfully achieved within 100 s.

Microchip capillary electrophoresis with electrochemical detection.

A novel microchip capillary electrophoresis system with electrochemical detection, using the replaceable microelectrode, is first reported. This kind of electrode can be fabricated in general laboratories and can be replaced quickly with electrodes of different materials according to the requirements of experiments. The end-column electrochemical detection on microchip CE was achieved by fixing the working electrode (such as carbon fiber, Pt, or Au, etc.) through a guide tube on the end of the separation channel. The experiment results indicate that the alignment of the electrode with the channel outlet can be carried out accurately and reproducibly, and therefore, the detection device has low noise and good reproducibility. The detection limit of dopamine is 2.4 x 10(-7) M, which is the lowest result reported so far. The separation and detection of dopamine, 5-hydroxytryptamine and epinephrine using carbon fiber and Pt microdisk electrodes within 50 s was successfully performed.

Highly sensitive chemiluminescence detection of copper(II) in capillary electrophoresis with field-amplified sample injection.

Field-amplified sample injection of copper(II) was investigated using capillary electrophoresis with chemiluminescence detection. The sensitivity of copper(II) has been improved markedly by the field-amplified sample injection technique and the detection limit reaches 2 x 10(-11) M. By injection of a short plug of water before sample introduction, the sensitivity can be further improved 5-fold and the detection limit reaches 4 x 10(-12) M. The relative standard deviations (n = 6) of the migration time and the peak height are 0.61% and 4.7% at 1.0 x 10(-9) M Cu(II), respectively. Parameters affecting the field-amplified sample injection, such as separation voltage and concentration of electrophoretic buffer, have been investigated.