Sheath Flow Cell Research Articles

2P-098 新しいさや流セルを使用した蛋白質一分子の折り畳み軌跡の観測(蛋白質・物性(2),第46回日本生物物理学会年会)

2P-098 新しいさや流セルを使用した蛋白質一分子の折り畳み軌跡の観測(蛋白質・物性(2),第46回日本生物物理学会年会)

Sheath-flow Fourier transform infrared spectrometry for the simultaneous determination of citric, malic and tartaric acids in soft drinks

A new approach for the determination of organic acids in soft drinks based on pH modulation and Fourier transform infrared spectroscopic detection is presented. The analytical readout was taken from the spectral changes induced by the pH change of the sample. The pH modulation was carried out in a novel sheath-flow cell which was connected to a sequential injection analysis system. The sheath-flow cell comprised three stream lines flowing adjacent to each other in a strongly laminar fashion. The sample (pH∼9) was introduced in the central channel whereas reagent (hydrochloric acid) was introduced in the outer channels. As a consequence of the laminar flow profile, hardly any mixing between the stream lines was observed while maintaining the flow, hence allowing the measurement of the sample spectrum at alkaline pH (∼9). Upon stopping the flow, diffusion of protons from the outer stream lines into the central line occurred resulting in a complete protonation of the analyte (pH∼2). The spectral changes were calculated and the region between 1400 and 1180 cm −1 used to set-up a partial least squares (PLS) calibration model. For the PLS model only standards containing the analytes but no matrix molecules were used. By evaluation of the spectral changes induced by the pH modulation the PLS model could successfully be applied to test samples containing sugars as well as to natural soft drinks.

A SHEATH FLOW CELL-BASED LASER INDUCED FLUORESCENCE DETECTION SYSTEM WITH PRE-IMAGING SPATIAL FILTERING FOR REDUCED DIMENSION SEPARATION TECHNIQUES

The construction, performance, and practical implementation of a rugged device for laser induced fluorescence (LIF) detection in capillary electrophoresis (CE) and liquid chromatography (LC) techniques is described. This device is based on a sheath flow cell (SFC) arrangement with attributes that include ruggedness, simplicity, ease of use, low cost, intra-day reproducibility, and small size. With this design, a pre-imaging mode of spatial filtering is employed to effectively reduce the contributions of both wall scatter and wall fluorescence to the background signal levels. A comparison of the pre-imaging spatial filtering and traditional methods of far-field spatial filtering is performed. This SFC system has been designed specifically for use in the UV spectral region, which can be very advantageous since it broadens the scope of the possible applications of LIF; however, fluorescence of the flow cell walls is particularly problematic in the UV region. The effect of flow cell geometry, slit widths, and detector position on fluorescent background levels are shown. The device has been applied to the fluorescence detection of three dansyl amino acids for both CE and capillary LC techniques. Efficiencies on the order of >105 plates/meter and 104 plates/meter are shown for CE and capillary LC techniques respectively. A comparison is made with CE between on-column and post-column detection demonstrating the latter preserves the integrity of a chromatographic separation.

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.

Independent optimization of capillary electrophoresis separation and native fluorescence detection conditions for indolamine and catecholamine measurements.

Separation conditions in capillary electrophoresis with native fluorescence detection often represent a compromise in terms of the separation and detection figures of merit. As both the separation and fluorescence properties greatly depend on pH, the ability to independently optimize pH in the separation capillary and the detection region can improve many complex separations. When using a sheath flow cell, the pH at the detection zone can be adjusted independently of the electrophoresis buffer pH. Using capillary electrophoresis with 257-nm excitation and native fluorescence detection, more than an order of magnitude improvement in the limits of detection for dopamine (from 1400 to 120 nM) and epinephrine (from 850 to 60 nM) is achieved by maintaining the basic separation conditions and an acidified sheath buffer. The detection of dopamine in an individual Aplysia californica cerebral ganglion neuron is demonstrated.

Fluorescence detection of proteins and amino acids in capillary electrophoresis using a post-column sheath flow reactor

A simple laser-induced fluorescence detection method for proteins and amino acids in capillary electrophoresis is reported. A sheath flow cell is utilized as a post-column reactor for fluorescence derivatization of proteins and amino acids by addition of o-phthaldialdehyde–2-mercaptoethanol to the sheath fluid. With the use of a 50 μm I.D. capillary, the limits of detection for carbonic anhydrase are 0.73 n M or 1.8 amol which represents a five- and two-fold improvement, respectively, over the best results previously reported for post-column detection. In addition, separation efficiencies up to 8.07·10 5 are achieved, and the detector response is linear over three-orders of magnitude. These results demonstrate that mixing is adequate and the reaction kinetics are rapid enough to provide sensitive detection with this approach. Also, because this post-column derivatization scheme requires no instrumental changes to a typical sheath flow cell detector, the system can be used for detection of pre-column labeled analytes and for native fluorescence detection.

Evaluation of a Sheath Flow Cuvette for Postcolumn Fluorescence Derivatization of DNA Fragments Separated by Capillary Electrophoresis

The investigation and evaluation of the sheath flow cell as a reaction chamber to postcolumn fluorescently derivatize DNA fragments separated by capillary electrophoresis is described herein. Use of the sheath flow cell arrangement facilitates the mixing of the intercalating dye, ethidium bromide (EB), and the effluent from the separation capillary by diffusion without a high degree of band dispersion. Theoretical plate counts of >1 × 10(6) are reported with the postcolumn derivatization technique, and resolution of all of the fragments in a φx-174-HaeIII digest is achieved. Optimization of experimental parameters such as flow rate, position of the detection zone, and EB concentration is examined. A limit of detection in the low nanograms-per-milliliter range with a linear dynamic range over 3 orders of magnitude is reported for a sample of φx-174-HaeIII digest. Evaluation of postcolumn derivatization for the investigation of DNA-protein interactions is demonstrated. The integrity of a DNA-trp-repressor protein interaction is maintained with the postcolumn approach but is compromised when EB is added to the running buffer.

High resolution multichannel fluorescence detection for capillary electrophoresis Application to multicomponent analysis

A wavelength-resolved fluorescence detector for laser-induced fluorescence detection in capillary electrophoresis (CE) is described that uses a charge injection device (CID) array detector. Post-column fluorescence detection occurs using a sheath flow cell. The limit of detection for fluorescein is 4.8·10 −11 M (29 000 molecules), the spectral resolution is 0.56 nm/pixel, and the spectrograph/CID monitors a 250 nm spectrum throughout the 250–875 nm range. Custom array readout, data manipulation and data processing methods are described to convert wavelength/spatial CID images into electropherograms. The application of the system to characterizing bilirubins in human serum is described, demonstrating the ability to match electrophoretic peaks to standards using spectral information.

Characterizing submicron vesicles with wavelength-resolved fluorescence in flow cytometry

Individual synthetic vesicles 0.1-1.0 micron in diameter are sized by using single-particle fluorescence emission spectra obtained in a custom sheath flow cell with an imaging spectrograph and a charge-coupled device. Data are acquired at 1 Hz, with limits of detection (3 sigma) less than 6.0 x 10(3) and 1.0 x 10(4) molecules of free sulforhodamine 101 and fluorescein, respectively, and with a spectral range for fluorescence emission collection from 350 to 800 nm (0.45 nm/pixel resolution). The system is used for small-particle population analysis by analyzing a suspension of submicron, unilamellar, synthetic vesicles prepared by standard procedures with phosphatidylserine and Texas red- or fluorescein head-group-conjugated dihydropalmitoylphosphatidylethanolamine. The submicron particles are individually identified, sized, and discriminated based on single-particle fluorescence emission spectra. Excellent agreement is found between fluorescence sizing data and transmission electron microscopic measurements.

Native fluorescence detection and spectral differentiation of peptides containing tryptophan and tyrosine in capillary electrophoresis

A native fluorescence detection system for capillary electrophoresis is described that achieves low attomole detection limits and simultaneous acquisition of complete fluorescence emission spectra. The system is designed for detection of peptides through the intrinsic fluorescence of tryptophan and tyrosine residues. The detection system employs a frequency doubled krypton laser operating at 284 nm for excitation, a sheath flow cell, a reflective f/1.2 microscope objective, an imaging spectrograph, and a CCD detector. The detection capabilities are characterized with tryptophan and tyrosine, which have limits of detection (3 sigma) of 2 x 10(-10) and, 2 x 10(-8) M, respectively. Acquisition of the fluorescence emission spectrum provides the ability to distinguish three classes of peptides: those that contain tryptophan, those that contain tyrosine, and those that contain both tryptophan and tyrosine.

Subattomole detection of amino acids by capillary electrophoresis based on semiconductor laser fluorescence detection

A pyronin succinimidyl ester fluorescent in the deep-red region is synthesized, whose absorption maximum and molar absorptivity are 663 nm and 8.2 × 10 4, respectively. This compound is used for fluorescence labeling of amino acids such as alanine, arginine, glycine, glutamic acid, and aspartic acid. The sample containing the labeled amino acids is separated by capillary electrophoresis and is detected by semiconductor laser fluorometry. A sheath flow cell is used for reduction of the light scatter from the cell wall. This compact and inexpensive analytical system allows detection of 0.8-4.5 attomole of amino acids.

The performance of some cell designs for laser-induced fluorescence detection in open-tubular liquid chromatography

The applicability of fluorescence detection in miniaturized cells using a helium—cadmium laser as the excitation source at 325 nm to open-tubular liquid chromatography has been studied. The investigated cells were fused-silica capillaries of 25 and 10 μm I.D., comprising part of the column or being coupled separately to it, and a sheath flow cell. The volume standard deviation of the various cells was found to be 0.1–2 nl which makes them suitable for the above purpose. The mass detection limits were found to be in the attogram range. However, the concentration detection limits, were found to be one to two orders of magnitude higher than those obtainable with conventional sized fluorescence detectors. This is mainly caused by the instability of the laser.