Portable capillary electrophoresis system with potential gradient detection for separation of DNA fragments

A portable capillary electrophoresis (CE) system with a novel potential gradient detection (PGD) was utilized to separate DNA fragments. For the first time it was demonstrated that separation of DNA fragments in polymer solution could be detected by a portable CE system integrated with PGD, with a limit of detection (LOD) comparable to that of the CE-ultraviolet (UV) method. Effects of buffer solution, sieving medium, and applied voltage were also investigated. The portable CE-PGD system shows several potential advantages, such as simplicity, cost effectiveness, and miniaturization.     

Rapid separations of nile blue stained microorganisms as cationic charged species by chip-CE with LIF

Rapid detection of microorganisms by alternative methods is desirable. Electromigration separation methods have the capability to separate microorganisms according to their charge and size and laser-induced fluorescence (LIF) detection have single-cell detection capability. In this work, a new combined separation and detection scheme was introduced using chip-based capillary electrophoresis (chip-CE) platform with LIF detection. Three microorganisms Escherichia coli, Staphylococcus aureus, and Candida albicans were selected as representatives of Gram-positive bacteria, Gram-negative bacteria, and fungi. While their cells carry an overall negative charge in neutral to alkaline pH, staining them with nile blue (NB) provided highly sensitive LIF detection with excitation and emission wavelengths at 635 nm and 685 nm, respectively, and at the same time, the overall charge was converted to positive. Electrolyte pH and concentration of polyethylene oxide (PEO) significantly affected the resolution of the microorganisms. Their optimal separation in the 14 mm separation channel was achieved in less than 30 s (R(s) > 5.3) in an electrolyte consisting of 3.94 mM Tris, 0.56 mM boric acid, 0.013 mM ethylenediaminetetraacetic acid disodium salt dihydrate (pH 10.5), and 0.025% PEO, with injection/separation voltages of +1000/+1000 V. The separation mechanism is likely employing contributions to the overall cationic charge from both the prevalently anionic membrane proteins and the cationic NB. Importantly, the resulting cationic NB-stained cells exhibited excellent separation selectivity and efficiency of ～38000 theoretical plates for rapid separations within 30-40 s. The results indicate the potential of chip-CE for microbial analysis, which offers separations of a wide range of species with high efficiency, sensitivity, and throughput.     

Speciation analysis of inorganic arsenic by microchip capillary electrophoresis coupled with hydride generation atomic fluorescence spectrometry

A novel method for speciation analysis of inorganic arsenic was developed by on-line hyphenating microchip capillary electrophoresis (chip-CE) with hydride generation atomic fluorescence spectrometry (HG-AFS). Baseline separation of As(III) and As(V) was achieved within 54 s by the chip-CE in a 90 mm long channel at 2500 V using a mixture of 25 mmol l(-1) H3BO3 and 0.4 mmol l(-1) CTAB (pH 8.9) as electrolyte buffer. The precisions (RSD, n=5) ranged from 1.9 to 1.4% for migration time, 2.1 to 2.7% for peak area, and 1.8 to 2.3% for peak height for the two arsenic species at 3.0 mg l(-1) (as As) level. The detection limits (3sigma) for As(III) and As(V) based on peak height measurement were 76 and 112 microg l(-1) (as As), respectively. The recoveries of the spikes (1 mg l(-1) (as As) of As(III) and As(V)) in four locally collected water samples ranged from 93.7 to 106%.     

带有可更换半自准工作电极的集成化毛细管电泳安培检测芯片

自Woolley等首次报道集成于玻璃芯片上的微型毛细管电泳-安培检测(Chip-based capillary electrophoresis with amperometric detection,CE-AD)系统以来,CE-AD以其高效、高速、高灵敏度以及易微型化集成化等特点引起研究者的关注．在芯片上实现柱端安培检测可用直接制作在芯片上的喷(镀)膜工作电极,或采用外置的壁喷式电极。前者集成化程度高,后者的工作电极可以更换,大大提高了芯片的重复利用率。     

微流控芯片电解质介导连接在柱式电导检测

研制了一种新型在柱式微流控芯片电导检测装置,利用电解质介导连接分离样品和检测电极,避免了电极的污染和中毒.在芯片的分离通道上设有双T型通道和十字型通道,分别用于进样和检测.检测电极分别置于十字通道口两端的储液池中,电极与芯片相互独立,简化了实验装置,便于电极的更换和清洗.采用缓冲溶液作介导电解质,减小了因两者浓度或种类不同而导致的基线漂移.与非接触电导接触相比,本装置在较低的检测电压(2.5～4.0 V)和频率(700～1700 Hz)范围即可获得相对灵敏的信号.在15 mmol/L MES-His(pH 5.8)的缓冲体系下,K+与Na+的检出限分别为0.5和0.1 μmol/L.     

A thin cover glass chip for contactless conductivity detection in microchip capillary electrophoresis

A microfabricated thin glass chip for contactless conductivity detection in chip capillary electrophoresis is presented in this contribution. Injection and separation channels were photolithographed and chemically etched on the surface of substrate glass, which was bonded with a thin cover glass (100 μm) to construct a new microchip. The chip was placed over an independent contactless electrode plate. Owing to the thinness between channel and electrodes, comparatively low excitation voltage (20–110 V in V_p–p) and frequency (40–65 kHz) were suitable, and favorable signal could be obtained. This microchip capillary electrophoresis device was used in separation and detection of inorganic ions, amino acids and alkaloids in amoorcorn tree bark and golden thread in different buffer solutions. The detection limit of potassium ion was down to 10 μmol/L. The advantages of this microchip system exist in the relative independence between the microchip and the detection electrodes. It is convenient to the replacement of chip and other operations. Detection in different position of the channel would also be available.     

Fabrication and performance of a three-dimensionally adjustable device for the amperometric detection of microchip capillary electrophoresis

A microchip CE-amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an AD cell containing a one-dimensionally adjustable disk detection electrode in a Plexiglas holder. It facilitates the precise 3-D alignment between the channel outlet and the detection electrode without a complicated 3-D manipulator. The performance of this unique system was demonstrated by separating five aromatic amines (1,4-phenyldiamine, aniline, 2-methylaniline, 4-chloroaniline, and 1-naphthylamine) of environmental concern. Factors influencing their separation and detection processes were examined and optimized. The five analytes have been well separated within 140 s in a 74 cm long separation channel at a separation voltage of +2500 V using a 10 mM phosphate buffer (pH 3.5). Highly linear response is obtained for the five analytes over the range 20-200 microM with the detection limits ranging from 0.46 to 1.44 microM, respectively. The present system demonstrated long-term stability and reproducibility with RSDs of less than 5% for the peak current (n = 9). The new approach for the microchannel-electrode alignment should find a wide range of applications in CE, flowing injection analysis, and other microfluidic analysis systems.     

A three-dimensionally adjustable amperometric detector for microchip electrophoretic measurement of nitroaromatic pollutants

A microchip capillary electrophoresis (CE)–amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an amperometric detection cell containing a one-dimensionally adjustable disc detection electrode in a Plexiglas holder. It facilitates the precise three-dimensional alignment between the channel outlet and the detection electrode without a complicated three-dimensional manipulator. The performance of this unique system was demonstrated by separating four nitroaromatic pollutants (nitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, and p-nitrobenzene). Factors influencing their separation and detection processes were examined and optimised. The four analytes have been well-separated within 120 s in a 75 cm long separation channel at a separation voltage of +2000 V using an electrophoretic separation medium containing 15 mM borax and 15 mM sodium dodecyl sulfate (pH 9.2). Highly linear response is obtained for the four analytes over the range of 0–5 ppm with the detection limits ranging from 12 to 52 ppb. The present system demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n = 9). The new approach for the microchannel–electrode alignment should find a wide range of applications in other microfluidic analysis systems.     

Integrated capillary electrophoresis amperometric detection microchip with replaceable microdisk working electrode. II. Influence of channel cross-sectional area on the separation and detection of dopamine and catechol

The interference of separation high voltage with the electrochemical detection is a major challenge to the microchip capillary electrophoresis-electrochemical detection systems with end-channel detection mode. Using dopamine and catechol as model analytes, the influences of channel cross-sectional area and channel-to-electrode distance on the high-voltage interference, accordingly on the separation and detection performances of the microchip capillary electrophoresis-electrochemical detection system were investigated. With the increase of the channel cross-sectional area from 312 through 450-615 microm2, the apparent half-wave potentials of hydrodynamic voltammetry for dopamine at the field strength of 288 V/cm shifted positively from 285 through 330-400 mV. By using a chip with the smallest channel cross-section (312 microm2 with top width of 37.3 microm and depth of 8.9 microm) the residual high-voltage field in the detection cell was small, so that detection was conducted at a channel-to-electrode distance of 20 microm to achieve better performances of separation and detection.     

Fluorescence detection system for capillary separations utilizing a liquid core waveguide with an optical fibre-coupled compact spectrometer

A fluorescence detection system for capillary liquid separation methods is described. The system is based on a silica capillary coated with a low refractive index fluoropolymer Teflon AF that serves both as a separation channel and as a liquid core waveguide (LCW). A fibre-coupled laser excites separated analytes in a detection point and arising fluorescence is collected at one end of the LCW capillary into the other optical fibre which brings it to a compact charge-coupled device (CCD) array spectrometer installed in a desktop computer. No additional components such as focusing optics or filters are necessary. This system was used for detecting isoelectrically focused fluorescent low-molecular-mass pI (isoelectric point) markers and fluorescein isothiocyanate (FITC) labelled proteins. The ability of the system to acquire fluorescent spectra is also demonstrated.     

塑料芯片毛细管电泳电化学检测系统及其性能评价

近年来,高分子芯片毛细管电泳技术发展迅速,以聚甲基丙烯酸甲酯(PMMA)为代表的塑料电泳芯片由于其低廉的制作成本与良好的电渗性能,已经成为芯片电泳技术发展的一个重要方向,电化学检测具有灵敏度高、选择性好和易于微型化等优点,因此在塑料芯片电泳领域中具有较好的应用前景。     

Development of a new hybrid technique for rapid speciation analysis by directly interfacing a microfluidic chip-based capillary electrophoresis system to atomic fluorescence spectrometry

This paper represents the first study on direct interfacing of microfluidic chip-based capillary electrophoresis (chip-CE) to a sensitive and selective detector, atomic fluorescence spectrometry (AFS) for rapid speciation analysis. A volatile species generation technique was employed to convert the analytes from the chip-CE effluent into their respective volatile species. To facilitate the chip-CE effluent delivery and to provide the necessary medium for subsequent volatile species generation, diluted HCl solution was introduced on the chip as the makeup solution. The chip-CE-AFS interface was constructed on the basis of a concentric "tube-in-tube" design for introducing a KBH4 solution around the chip effluent as sheath flow and reductant for volatile species generation as well. The generated volatile species resulting from the reaction of the chip-CE effluent and the sheath flow were separated from the reaction mixture in a gas-liquid separator and swept into the AFS atomizer by an argon flow for AFS determination. Inorganic mercury (Hg(II)) and methylmercury (MeHg(I)) were chosen as the targets to demonstrate the performance of the present technique. Both mercury species were separated as their cysteine complexes within 64 s. The precision (relative standard deviation, RSD, n = 5) of migration time, peak area, and peak height for 2 mg.L(-1) Hg(II) and 4 mg.L(-1) MeHg(I) (as Hg) ranged from 0.7 to 0.9%, 2.1 to 2.9%, and 1.5 to 1.8%, respectively. The detection limit was 53 and 161 microg.L(-1) (as Hg) for Hg(II) and MeHg(I), respectively. The recoveries of the spikes of mercury species in four locally collected water samples ranged from 92 to 108%.     

Improvement of lipoprotein separation with a guard channel prior to asymmetrical flow field-flow fractionation using fluorescence detection

In this article, a simple experimental approach to improve lipoprotein separation and detection in flow field-flow fractionation (FlFFF) is detailed. Lipoproteins are globular particles composed of lipids and proteins in blood serum and their roles include transferring fats and cholesterols through blood vessels throughout the body. Especially, presence of small, dense low-density lipoproteins (LDL) is associated with cardiovascular risk. Two experimental approaches were explored in this study: an increase in the reproducibility of LDL particle separation by implementing a guard channel prior to an asymmetrical FlFFF (AFlFFF) channel in order to deplete small molecular weight serum proteins and reducing the required injection volume of a serum sample by implementing fluorescence detection. The guard channel was made of a simple hollow fiber module so that the serum sample can be washed with the help of radial flow prior to injection into the AFlFFF channel. The channel was tested with protein standards and serum samples to ensure precision of the retention time and the protein recovery rate. A fluorescent phospholipid dye was utilized to label lipoprotein particles before separation for fluorescence detection, which resulted in a reduction of the required injection volume of serum.     

Microchip electrophoresis coupled with on-line magnetic separation and chemiluminescence detection for multiplexed immunoassay

A facile and universal strategy for multiplexed immunoassay is proposed. The strategy is based on microchip electrophoresis (MCE) coupled with on-line magnetic separation and chemiluminescence (CL) detection. The system consisted of a microchip, an electromagnet, and a photomultiplier. The realization of multiplexed immunoassay protocol involves sampling magnetic nanoparticles (MNPs) labeled antibodies, N-(4-aminobutyl)-N-ethyl-isoluminol (ABEI) labeled antigens and free antigens in the precolumn reactor, on-line immunoreaction, capturing the MNPs-immunocomplexes, and the separation of unconjugated ABEI-labeled antigens. After on-line magnetic separation, the free ABEI-labeled antigens were transported into the separation channel, and mixed with hydrogen peroxide (H(2) O(2) ) in the presence of horseradish peroxidase in the postcolumn reactor, and producing CL emission. Using this arrangement, multiple analytes could be measured simultaneously by performing the technical operations for a single assay. As a proof-of-concept, the multiplexed immunoassay was evaluated for the simultaneous determination of five model analytes (i.e. hydrocortisone, corticosterone, digoxin, testosterone, and estriol). The results exhibited excellent precision and sensitivity, the relative standard deviations for nine times detection were lower than 4.7% for all the five components, and the detection limits of five analytes were in the range of 3.6-4.9 nM. The MCE system was validated using two human serum-based control samples containing five analytes.     

Microchip with an open tubular immobilized ph gradient for UV whole column imaging detection

This study reports a new method for establishing an open tubular IPG in a microchip coupled with a whole column image detection (WCID) system for protein separation applications. This method allows a wider range of immobilized pH (2.6–9.5) to be established in a PDMS/quartz channel by controlling the diffusion of acidic and basic polymer solutions into the channel through well‐designed channel dimensions. The developed pH gradient was experimentally validated by performing the separation of a mixture of standard pI markers. It was further validated by the separation of the hemoglobin control AFSC sample. This method is advantageous over existing IPG methods because it has a wider range of pH and maintains the open tubular feature that matches the UV WCID to improve the sensitivity.     

Separation and electrochemical detection of paracetamol and 4-aminophenol in a paper-based microfluidic device

The present work describes the construction and application of a simple, low cost and sensitive microfluidic paper-based device with electrochemical detection for the detection of paracetamol and 4-aminophenol. The separation channels of a width of 2.0 mm were created on paper using a wax printing process to define the regions of the device. A baseline separation level of the analytes can be obtained in 0.1 mol L⁻¹ acetate buffer solution at pH 4.5 and by injecting 500 nL of the standard solutions at 12 mm from the working electrode. The electrochemical detection system was created at the end of the channels through a process known as sputtering. The previously separated analytes were detected at the end of the hydrophilic separation channel by applying a potential of 400 mV vs. pseudo Au on the working electrode. Experimental variables such as type of paper (cation exchanger and n1), pH, sample volume, applied potential and distance of sample injection were evaluated and, under the conditions of higher response, it was possible to obtain detection limits of 25.0 and 10.0 μmol L⁻¹ for paracetamol and 4-aminophenol, respectively.     

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.     

Microchip capillary electrophoresis with electrochemical detector for fast measurements of aromatic amino acids

A method based on microchip capillary electrophoresis with amperometric detection was developed for the rapid separation and direct detection of oxidizable aromatic amino acids (without prior derivatization). The working electrode was a thick-film carbon strip electrode positioned opposite the outlet of the separation channel. Factors influencing the separation and detection processes were examined and optimized. The five aromatic amino acids, tyrosine, 5-hydroxytryptophan, tryptophan, p-aminobenzoic acid, and m-aminobenzoic acid, can be well separated within 5 min using a separation voltage of 2000 V and a 25 mM phosphate buffer (pH 7.0) run buffer containing 50 mM sodium dodecylsulfate. Most favorable amperometric detection was obtained at +0.95 V. Linear calibration plots are observed for micromolar concentrations of the oxidizable amino acids. The new protocol offers good stability and for reproducibility, with relative S.D. of less than 5% for both migration times and peak currents (n=8). It should be useful for the analysis of aromatic amino acids, as desired for life sciences.     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.     

In-channel indirect amperometric detection of nonelectroactive anions for electrophoresis on a poly(dimethylsiloxane) microchip

In the present paper, we describe a microfluidics-based sensing system for nonelectroactive anions under negative separation electric field by mounting a single carbon fiber disk working electrode (WE) in the end part of a poly(dimethylsiloxane) microchannel. In contrast to work in a positive separation electric field described in our previous paper (Anal. Chem. 2004, 76, 6902-6907), here the electrochemical reduction reaction at the WE is not coupled with the separation high-voltage (HV) system, whereas the electrochemical oxidation reaction at the WE is coupled with the separation HV system. The electroactive indicator is the carbon fiber WE itself but not dissolved oxygen. This provides a convenient and sensitive means for the determination of nonelectroactive anions by amperometry. The influences of separation voltage, detection potential, and the distance between the WE and the separation channel outlet on the response of the detector have been investigated. The present detection mode is successfully used to electrochemically detect F-, Cl-, SO4(2-), CH3COO-, H2PO4-. Based on the preliminary results, a detection limit of 2 microM and a dynamic range up to three orders of magnitude for Cl- could be achieved.