Microfabricated capillary electrophoresis amino acid chirality analyzer for extraterrestrial exploration.

Chiral separations of fluorescein isothiocyanate-labeled amino acids have been performed on a microfabricated capillary electrophoresis chip to explore the feasibility of using such devices to analyze for extinct or extant life signs in extraterrestrial environments. The test system consists of a folded electrophoresis channel (19.0 cm long x 150 microns wide x 20 microns deep) that was photolithographically fabricated in a 10-cm-diameter glass wafer sandwich, coupled to a laser-excited confocal fluorescence detection apparatus providing subattomole sensitivity. Using a sodium dodecyl sulfate/gamma-cyclodextrin pH 10.0 carbonate electrophoresis buffer and a separation voltage of 550 V/cm at 10 degrees C, baseline resolution was observed for Val, Ala, Glu, and Asp enantiomers and Gly in only 4 min. Enantiomeric ratios were determined for amino acids extracted from the Murchison meteorite, and these values closely matched values determined by HPLC. These results demonstrate the feasibility of using microfabricated lab-on-a-chip systems to analyze extraterrestrial samples for amino acids.     

Microfabricated 384-lane capillary array electrophoresis bioanalyzer for ultrahigh-throughput genetic analysis

A microfabricated 384-lane capillary array electrophoresis device is developed and utilized for massively parallel genetic analysis. The 384 capillary lanes, arrayed radially about the center of a 200-mm-diameter glass substrate sandwich, are constructed using scalable microfabrication techniques derived from the semiconductor industry. Samples are loaded into reservoirs on the perimeter of the wafer, separated on the 8-cm-long poly(dimethylacrylamide) gel-filled channels, and detected with a four-color rotary confocal fluorescence scanner. The performance and throughput of this bioanalyzer are demonstrated by simultaneous genotyping 384 individuals for the common hemochromatosis-linked H63D mutation in the human HFE gene in only 325 s. This lab-on-a-chip device thoroughly exploits the power of microfabrication to produce high-density capillary electrophoresis arrays and to use them for high-throughput bioanalysis.     

CMOS absorbance detection system for capillary electrophoresis

This paper presents a cost-effective portable photodetection system for capillary electrophoresis absorptiometry. By using a CMOS BDJ (buried double p–n junction) detector, a dual-wavelength method for absorbance measurement is implemented. This system includes associated electronics for low-noise pre-amplification and A/D conversion, followed by digital signal acquisition and processing. Two signal processing approaches are adopted to enhance the signal to noise ratio. One is variable time synchronous detection, which optimizes the sensitivity and measuring rate compared to a conventional synchronous detection technique. The other is a statistical approach based on principal component analysis, which allows optimal estimation of detected signal. This system has been designed and tested in capillary electrophoresis conditions. Its operation has been verified with performances comparable to those of a commercialized spectrophotometric system (HP-3D CE). With potential on-chip integration of associated electronics, it may be operated as an integrable detection module for microchip electrophoresis and other microanalysis systems.     

Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary electrophoresis laser-induced fluorescence analysis

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes whose function in specific aminoacylation of tRNAs is central to the process of protein translation, which occurs in the cytoplasm of all living cells. In addition to their well-established cytoplasmic localization, fluorescence microscopy studies and analysis of the aminoacylation state of nuclear tRNAs have revealed that synthetases are localized in the nuclei of cells from several species including Xenopus laevis and Saccharomyces cerevisiae. Whether nuclear localization of aaRSs is a general phenomenon that occurs in all eukaryotic cells is an open question. In the work described here, human methionyl-tRNA synthetase (MRS) and human lysyl-tRNA synthetase (KRS) were expressed in human-derived DeltaH2-1 osteosarcoma cells as enhanced green fluorescent protein (EGFP) fusion proteins. The subcellular localization of these EGFP-aaRSs was first probed by fluorescence microscopy using cells that coexpressed EGFP-aaRS and a nuclear marker fusion protein, nuDsRed. As expected, both aaRSs were present in the cytosol, while only EGFP-MRS was also clearly localized in the nucleus. To confirm these findings, and to investigate a potentially more sensitive, general method for nuclear localization studies, capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze single DeltaH2-1 cells expressing both EGFP-aaRS and nuDsRed. While cytosolic EGFP signals were detected for both EGFP-MRS and EGFP-KRS, only EGFP-MRS was found in the nucleus, along with nuDsRed. The detection of EGFP-MRS in nuclei of DeltaH2-1 cells demonstrates the feasibility of using CE-LIF analysis in nuclear localization studies of proteins in mammalian cells.     

Microfabricated electrophoresis chip for bioassay of renal markers

A novel capillary electrophoresis chip-based detection system for simultaneous measurements of the renal markers creatine, creatinine, p-aminohippuric acid, and uric acid is described. Fluid control is used for mixing the sample with the enzymes creatininase (CA), creatinase (CI), and sarcosine oxidase (SOx) and for separating the neutral hydrogen peroxide end product from the anionic p-aminohippuric and urate species. The 'total' (creatinine and creatine) signal was measured with the running buffer containing all three enzymes, while the creatine signal alone was recorded by mixing the sample with the CI-SOx solution. Creatinine concentrations are measured by comparing the response in the presence and absence of CA. The peroxide product and the oxidizable p-aminohippuric and uric acids are detected electrochemically at a downstream gold-coated thick-film amperometric detector. The four renal markers are readily measured within 5 min, while creatinine/creatine within less than 2 min. Factors influencing the performance, including the level of three enzymes, separation voltage, and detection potential, are optimized. Applicability to urine samples is demonstrated. Such a multianalyte microchip detection device would allow renal function testing to be performed more rapidly, easily, and economically in the point-of-care setting.     

Microfabricated devices for capillary electrophoresis-electrospray mass spectrometry.

Two fundamental approaches for the coupling of microfabricated devices to electrospray mass spectrometry (ESI-MS) have been developed and evaluated. The microdevices, designed for electrophoretic separation, were constructed from glass by standard photolithographic/wet chemical etching techniques. Both approaches integrated sample inlet ports, preconcentration sample loops, the separation channel, and a port for ESI coupling. In one design, a modular, reusable microdevice was coupled to an external subatmospheric electrospray interface using a liquid junction and a fused silica transfer capillary. The transfer capillary allowed the use of an independent electrospray interface as well as fiber optic UV detection. In the second design, a miniaturized pneumatic nebulizer was fabricated as an integral part of the chip, resulting in a very simple device. The on-chip pneumatic nebulizer provided control of the flow of the electrosprayed liquid and minimized the dead volume associated with droplet formation at the electrospray exit port. Thus, the microdevice substituted for a capillary electrophoresis instrument and an electrospray interface--traditionally two independent components. This type of microdevice is simple to fabricate and may thus be developed either as a part of a reusable system or as a disposable cartridge. Both devices were tested on CE separations of angiotensin peptides and a cytochrome c tryptic digest. Several electrolyte systems including a transient isotachophoretic preconcentration step were tested for separation and analysis by an ion trap mass spectrometer.     

Microfabricated two-dimensional electrophoresis device for differential protein expression profiling

A microfluidic separation system is developed to perform two-dimensional differential gel electrophoretic (DIGE) separations of complex, cellular protein mixtures produced by induced protein expression in E. coli. The micro-DIGE analyzer is a two-layer borosilicate glass microdevice consisting of a single 3.75 cm long channel for isoelectric focusing, which is sampled in parallel by 20 channels effecting a second-dimension separation by native electrophoresis. The connection between the orthogonal separation systems is accomplished by smaller channels comprising a microfluidic interface (MFI) that prevents media leakage between the two dimensions and enables facile loading of discontinuous gel systems in each dimension. Proteins are covalently labeled with Cy2 and Cy3 DIGE and detected simultaneously with a rotary confocal fluorescence scanner. Reproducible two-dimensional separations of both purified proteins and complex protein mixtures are performed with minimal run-to-run variation by including 7 M urea in the second-dimension separation matrix. The capabilities of the micro-DIGE analyzer are demonstrated by following the induced expression of maltose binding protein in E. coli. Although the absence of sodium dodecyl sulfate (SDS) in the second-dimension sizing separation limits the orthogonality and peak capacity of the separation, this analyzer is a significant first step toward the reproducible two-dimensional analysis of complex protein samples in microfabricated devices. Furthermore, the microchannel interface structures developed here will facilitate other multidimensional separations in microdevices.     

Identification of proteins in single-cell capillary electrophoresis fingerprints based on comigration with standard proteins

In the previous paper in this Journal, we reported the use of capillary sieving electrophoresis to characterize proteins expressed by single cancer cells at specific phases in the cell cycle. Analysis of the data revealed one component with cell cycle-dependent changes in expression at the 99% confidence limit. However, the amount of protein present in a single cell is far too small to allow its direct identification by mass spectrometry. In this paper, we report a method by which such proteins can be tentatively identified. We perform standard SDS-PAGE electrophoresis of the proteins contained within a homogenate prepared from an HT29 cell culture. Proteins extracted from bands in the gel are identified by mass spectrometry. The proteins also provide a set of standards that can be used to spike the sample before capillary sieving electrophoresis (CSE) separation; comigration is taken as evidence for the identity of the target protein. In a proof-of-principle experiment, a single band migrating at approximately 47 kDa was isolated from the SDS-PAGE gel generated from the HT29 cell line. Proteins extracted from this band were used to spike a CSE separation of the same extract. This band comigrated with a cell cycle-dependent component identified from single-cell analysis. In-gel digestion and LC/MS/MS were used to identify five proteins, including cytokeratin 18, which is the product of the most highly expressed gene in this cell line.     

Nanoinjector for capillary electrophoresis and capillary electrochromatography

A rotary valve nanoinjector was devised for use in capillary electrophoresis (CE) and capillary electrochromatography (CEC). A fused-silica capillary tip was inserted in a small through-hole in the rotor. The narrow and short capillary tip, with an inner volume of 6-24 nL, was embedded in the hole using epoxy resin. The injection volume was confirmed chromatographically by comparing the peak areas obtained with the nanoinjector to those of a conventional injector. In addition, both the rotor and stator of the injector were made of a nonconducting material, polyimide resin, to be utilized for CE and CEC. The application of the nanoinjector for CE was demonstrated.     

Dual conductivity/amperometric detection system for microchip capillary electrophoresis

The performance characteristics and advantages of a new dual electrochemical microchip detection system based on simultaneous conductivity and amperometric measurements are described. The system relies on the combination of a contactless conductivity detector with an end-column thick-film amperometric detector. Such coupling of the conductivity and amperometric detection modes in a single separation channel greatly enhances the sample characterization to offer simultaneous measurements of both ionic and electroactive species, improved reproducibility, and confirmation of peak identity. The simultaneous measurement of nitroaromatic and ionic explosives is used for demonstrating the ability to detect both electroactive and ionic species. Major improvements are also observed for analytes responding at both detectors. For example, the generation of dual response ratios can be used to improve the reproducibility and confirm the peak identity/integrity. Such dual response ratios reflect the distinct redox and conductivity properties of the individual analytes. The independence of the two detectors is reflected in the absence of "cross-talk" effects. The behavior of the dual detector is comparable with those of the individual detectors. Such a dual electrochemical detection system is easy to implement and requires inherently portable low-cost instrumentation.     

Chip-capillary hybrid device for automated transfer of sample preseparated by capillary isoelectric focusing to parallel capillary gel electrophoresis for two-dimensional protein separation

In this article, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to reroute the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed.     

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.     

In-line valve injection for capillary electrophoresis

Direct in-line injection is successfully demonstrated for capillary electrophoresis using a commercially available injection valve designed for liquid chromatographic applications. The internal, fluid-contacting materials in this valve injector are composed of ceramics and PEEK (polyetheretherketone). In studies up to 20 kV, this materials design provides a sufficient dielectric interface to insulate the high-voltage buffer from the metal valve body. Partial-loop injections from 6 to > 60 nL are shown to be highly reproducible and generally consistent with direct electrokinetic injections under the same experimental conditions. The small extracolumn variance contributed by the valve injection system is symmetrical, and the measured theoretical plates for 75-microm- and 100-microm-i.d. separation capillaries are 1.6 x 10(5) and 2.5 x 10(5), respectively. As a result, the separation performance is quite good, demonstrating the viability of in-line valve injection for capillary electrophoresis. This development in capillary electrophoretic instrumentation has important implications for the advancement of electrophoretic applications as well as for the design of completely integrated analysis systems.     

Passive conductivity detection for capillary electrophoresis

A passive electrochemical detection principle that can be applied to capillary electrophoresis is presented. The separation electrical field is used to generate a potential difference between two electrodes located along the channel. For constant-current electrophoresis, the generated signal is proportional to the resistance of the solution passing between the two electrodes. Contrary to conductivity detectors that are ac driven and need to be decoupled from the separation field, the passive detection directly takes advantage of the separation field. The signal is simply measured by a high-impedance voltmeter. The detection concept has been validated by numerical simulations showing how the magnitude of the signal is related to the ratio between the electrode distance and the length of the sample plug. As a proof of the principle, this detection concept has been demonstrated by the electrophoretic separation of three alkali ions on a polymer microchip. Based on preliminary results, a detection limit of 20 microM and a dynamic range of up to 3 orders of magnitude have been achieved.     

Contactless conductivity detection for capillary electrophoresis

A contactless capacitively coupled conductivity detector for capillary electrophoresis is introduced. The detector consists of two electrodes which are placed cylindrically around the outer polyimide coating of the fused-silica capillary with a detection gap of 2 mm. The electrodes form a cylindrical capacitor, and the electric conductivity of the solution in the gap between the electrodes is measured. A high audio or low ultrasonic frequency for coupling of the ac voltage is used in order to minimize the influence of reactance of the liquid. For an improved version of the detector, two syringe cannulas are used as the electrodes and the capillary is simply assembled into the tubing. This allows an easy placement of the detector on various positions along the capillary. The limit of detection of inorganic cations and anions is 200 ppb, as determined for sodium and chloride, respectively.     

Fluorescence correlation spectroscopy for rapid multicomponent analysis in a capillary electrophoresis system

We describe a new technique for performing multicomponent analysis using a combination of capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS), which we refer to as CE/FCS. FCS is a highly sensitive and rapid optical technique that is often used to perform multicomponent analysis in static solutions based on the different diffusion times of the analyte species through the detection region of a tightly focused laser beam. In CE/FCS, transit times are measured for a mixture of analytes continuously flowing through a microcapillary in the presence of an electric field. Application of an electric field between the inlet and outlet of the capillary alters the transit times, depending on the magnitude and polarity of the applied field and the electrophoretic mobilities of the analytes. Multicomponent analysis is accomplished without the need to perform a chemical separation, due to the different electrophoretic mobilities of the analytes. This technique is particularly applicable to ultradilute solutions of analyte. We have used CE/FCS to analyze subnanomolar aqueous solutions containing mixtures of Rhodamine 6G (R6G) and R6G-labeled deoxycytosine triphosphate nucleotides. Under these conditions, fewer than two molecules were typically present in the detection region at a time. The relative concentrations of the analytes were determined with uncertainties of ～10%. Like diffusional FCS, this technique is highly sensitive and rapid. Concentration detection limits are below 10(-)(11) M, and analysis times are tens of seconds or less. However, CE/FCS does not require the diffusion coefficients of the analytes to be significantly different and can, therefore, be applied to multicomponent analysis of systems that would be difficult or impossible to study by diffusional FCS.     

Using nonequilibrium capillary electrophoresis of equilibrium mixtures for the determination of temperature in capillary electrophoresis

Until now, all methods for temperature sensing in capillary electrophoresis (CE) relied on molecular probes with temperature-dependent spectral/optical properties. Here we introduce a nonspectroscopic approach to determining temperature in CE. It is based on measuring a temperature-dependent rate constant of complex dissociation by means of a kinetic CE method known as nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). Conceptually, a calibration curve of "the rate constant versus temperature" is built using NECEEM and a CE instrument with a reliable temperature control or, alternatively, a nonelectrophoretic method, such as surface plasmon resonance. The calibration curve is then used to find the temperature during CE in the same buffer but with another CE apparatus or under otherwise different conditions (cooling efficiency, length and diameter of the capillary, electrical field, etc.). In this proof-of-principle work, we used the dissociation of a protein-DNA complex to demonstrate that the NECEEM-based temperature determination method allows for temperature determination in CE with a precision of 2 degrees C. Then, we applied the NECEEM-based temperature determination method to study heat dissipation efficiency in CE instruments with active and passive cooling of the capillary. The nonspectroscopic nature of the method makes it potentially applicable to nonspectroscopic detection schemes, e.g. electrochemical detection. A "kinetic probe" can be coloaded into the capillary along with a sample for in situ temperature measurements. Higher order chemical reactions can also be used for temperature sensing, provided a kinetic CE method for measuring a corresponding rate constant is available.     

Field amplified separation in capillary electrophoresis: a capillary electrophoresis mode

In field-amplified injection in capillary electrophoresis (CE), the capillary is filled with two buffering zones of different ionic strength; this induces an amplified electrical field in the low ionic strength zone and a lower field in the high ionic strength zone, making sample stacking feasible. The electroosmotic flow (eof) usually observed in CE, however, displaces the low field zone and induces an extra band broadening preventing any CE separation in the field-amplified zone. These limitations have originated the restricted use of field amplification in CE only for stacking purposes. For the first time, in this work it is theoretically shown and experimentally corroborated that CE separation speed and efficiency can simultaneously be increased if the whole separation is performed in the field-amplified zone, using what we have called field amplified separation in capillary electrophoresis (FAsCE). The possibilities of this new CE mode are investigated using a new and simple coating able to provide near-zero eof at the selected separation pH. Using FAsCE, improvements of 20% for separation speed and 40% for efficiency are achieved. Moreover, a modified FAsCE approach is investigated filling the capillary with the high ionic strength buffer up to the interior of the detection window. Under these conditions, an additional 3-fold increase in sensitivity is also observed. The most interesting results were obtained combining the short-end injection mode and this modified FAsCE approach. Under these conditions, a part of a 3-fold improvement in efficiency and sensitivity, the total analysis time was drastically reduced to 40 s, giving rise to a time reduction of more than 7-fold compared to normal CE. This speed enhancement brings about one of the fastest CE separations achieved using capillaries, demonstrating the great possibilities of FAsCE as a new, sensitive, efficient, and fast CE separation mode.