On-column surface-enhanced Raman spectroscopy detection in capillary electrophoresis using running buffers containing silver colloidal solutions

Direct on-column surface-enhanced Raman spectroscopy (SERS) detection is demonstrated in capillary electrophoresis (CE). Distinctive SERS spectra of two test compounds, riboflavin and Rhodamine 6G, are obtained in 100 microm i.d. fused-silica capillaries under CE conditions using running buffers that contain silver colloidal solutions. Detection is performed using an unmodified commercial Raman spectrometer in a confocal microscope mode of operation. The effects of laser power, wavelength, spectra acquisition time, silver colloidal concentration, and applied voltage (i.e., flow rate) on the quality of SERS spectra are evaluated. Using laser powers of 17 mW (at the sample) at 515 nm and employing 1 s spectral acquisition times, spectra with bands exhibiting signal-to-noise ratios greater than 10 could be obtained for 1.0 x 10(-6) M riboflavin and very low nanomolar concentrations of Rhodamine 6G. This was accomplished without optimization of silver colloidal solution compositions and by using a low-throughput spectrometer. Incorporation of the colloidal solutions into running buffers is shown to have little effect on the separation of the test compounds as monitored using a laser-induced fluorescence instrumental scheme. However, SERS spectra degrade if the capillary is not rinsed between experiments. Riboflavin and Rhodamine 6G spectra are obtained on-the-fly for actual CE separations. In the case of the latter solute, the injected quantity was approximately 90 amol.     

High-throughput axial MALDI-TOF MS using a 2-kHz repetition rate laser

A high-throughput axial MALDI-TOF mass spectrometer utilizing a laser with a 2-kHz pulse repetition-rate was constructed and tested. This fast mass spectrometer provided a data acquisition rate 10 times faster than a commercially available (200 Hz) axial mass spectrometer, while maintaining comparable limits of detection (200 amol of Glu fib peptide). Mass resolution, only slightly less than the commercial instrument (10 000 vs 14 000), was sufficient for baseline resolution of isotopic clusters of peptides with m/z <2700. A new method of mass calibration, which combined a limited number of internal and external standards, provided the same 15 ppm mass accuracy over the entire sample plate on either instrument. Implementing the 2-kHz laser required a faster data acquisition system and high-voltage pulse electronics, together with a novel strategy for rapid sample plate movements during acquisition, to achieve a sample analysis rate of up to 2 spots/s (with 800 shots/spot). The overall performance of the fast MALDI-TOF MS instrument was demonstrated by the acquisition, in 12 min, of an LC-MS data set from a plate of 625 fractions collected during LC separation of an 16O/18O differentially labeled proteomic sample of a tryptic digest of an E. coli lysate.     

Attomole amino acid determination by capillary zone electrophoresis with thermooptical absorbance detection

Attomole quantities of 4-(dimethylamino)azobenzene-4'-sulfonyl chloride derivatized amino acids are separated by using capillary zone electrophoresis in a mixed acetonitrile/aqueous buffer system. Detection is performed with an on-column thermooptical absorbance detection technique based on a 130-mW argon ion pump laser. Detection limits for the concentration of analyte injected onto the column range from 5 x 10(-8) M for methionine to 5 x 10(-7) M for aspartic acid. Only 37 amol of methionine and 450 amol of aspartic acid are contained within the subnanoliter injection volume. It is interesting to note that these limits are a factor of 4 superior to the best fluorescence detection limit reported for chromatographic separation of amino acids. A subnanoliter sample of derivatized human urine was analyzed with this technique; quantities of amino acids contained within the sample are 3 orders of magnitude greater than the detection limit.     

High sensitivity Fourier transform ion cyclotron resonance mass spectrometry for biological analysis with nano-LC and microelectrospray ionization

Modifications to a 7 T nano-LC micro-ESI FT-ICR mass spectrometer, including a shorter octopole, approximately 100% duty cycle, improved nano-LC micro-ESI emitter tips, and reverse-phase HPLC resins that require no ion-pairing agent, combine to achieve attomole detection limit. Three peptides in a mixture totaling 500 attomoles (amol) each in water (10 microL, 50 amol/microL) are separated and detected, demonstrating detection from a mixture at low endogenous biological concentration. Two peptides in a mixture totaling 500 amol each in artificial cerebrospinal fluid (1 microL, 500 amol/microL) are separated and detected, demonstrating detection from a mixture at a biological concentration in a biological solvent. The highest sensitivity is attained with arg8-vasotocin, in which a total of 300 amol is detected in artificial cerebrospinal fluid (1 microL, 300 amol/microL) and a total of 100 amol in water (1 microL, 100 amol/microL). Arg8-vasotocin isolated from the pineal gland of rainbow trout is detected, demonstrating the ability of FT-ICR to detect and identify a true endogenous biological analyte.     

Analysis of carbonic anhydrase in human red Blood cells using capillary electrophoresis/ electrospray ionization-mass spectrometry

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of human red blood cells (RBCs) using the split-flow technique for interfacing CE to MS. By using a long (approximately125-cm) and narrow (approximately 15-microm-i.d.) capillary, the four major proteins of the RBC, which are hemoglobin (Hb, alpha- and beta-chains, 900 amol/chain), carbonic anhydrase I (CAI, approximately 7 amol/cell), and carbonic anhydrase II (CAII, approximately 0.8 amol/cell), were separated from each other and detected at low-attomole levels in one run and minimal sample preparation. Under these conditions, the detection limits for CAI and CAII in lysed RBCs were approximately 20 and approximately 44 amol, respectively. The approximately 20-amol detection limit of CAI was confirmed by the CE/ESI-MS analysis of three intact RBCs that had been drawn into the capillary under a microscope. A shorter capillary (approximately 55 cm long) provided faster analysis time but did not separate CAII from the beta-chain of hemoglobin, causing the CAII signal to be masked by the background chemical noise generated by the approximately 1,000 x molar excess of the beta-chain. Under this condition, the CAII detection limit increased to approximately 500 amol. From three methods of sample introduction (injection of lysed blood, injection of intact cells under microscope, and injection of intact cells suspended in saline solution), injection of lysed blood provided the optimum sensitivity. It was found that a background electrolyte (BGE) containing 0.1% acetic acid in water worked best for the analysis of intact cells, while a BGE containing 0.1% acetic acid in water + acetonitrile (50/50 by volume) worked best for the analysis of lysed blood.     

Online nanoflow reversed phase-strong anion exchange-reversed phase liquid chromatography-tandem mass spectrometry platform for efficient and in-depth proteome sequence analysis of complex organisms

The dynamic range of protein expression in complex organisms coupled with the stochastic nature of discovery-driven tandem mass spectrometry (MS/MS) analysis continues to impede comprehensive sequence analysis and often provides only limited information for low-abundance proteins. High-performance fractionation of proteins or peptides prior to mass spectrometry analysis can mitigate these effects, though achieving an optimal combination of automation, reproducibility, separation peak capacity, and sample yield remains a significant challenge. Here we demonstrate an automated nanoflow 3-D liquid chromatography (LC)-MS/MS platform based on high-pH reversed phase (RP), strong anion exchange (SAX), and low-pH reversed phase (RP) separation stages for analysis of complex proteomes. We observed that RP-SAX-RP outperformed RP-RP for analysis of tryptic peptides derived from Escherichia coli and enabled identification of proteins present at a level of 50 copies per cell in Saccharomyces cerevisiae, corresponding to an estimated detection limit of 500 amol, from 40 μg of total lysate on a low-resolution 3-D ion trap mass spectrometer. A similar study performed on a LTQ-Orbitrap yielded over 4000 unique proteins from 5 μg of total yeast lysate analyzed in a single, 101 fraction RP-SAX-RP LC-MS/MS acquisition, providing an estimated detection limit of 65 amol for proteins expressed at 50 copies per cell.     

Development of a simple algorithm for the detection of chilling injury in cucumbers from visible/near-infrared hyperspectral imaging

Hyperspectral images of cucumbers under a variety of conditions were acquired to explore the potential for the detection of chilling-induced damage in whole cucumbers. Region of interest (ROI) spectral features of chilling injured areas, resulting from chilling treatment at 0 degrees C, showed the reduction of reflectance intensity over the period at post-chilling room temperature (RT) storage. A large spectral difference between good, smooth skins and chilling-injured skins occurred in the 700-850 nm visible/near-infrared (NIR) region. Both simple band ratio algorithms and principal component analysis (PCA) were attempted to discriminate the ROI spectra of good cucumber skins from those of chilling injured ones. Results revealed that both the dual-band ratio algorithm (R(811nm)/R(756nm)) and a PCA model from a narrow spectral region of 733-848 nm can detect chilling-injured skins with a success rate of over 90%. The results also suggested that chilling injury is relatively difficult to detect at the initial post-chilling RT stage, especially during the first 0-2 days in storage, due to insignificant manifestation of chilling induced symptoms.     

A microchip electrophoresis device with integrated electrochemical detection: a direct comparison of constant potential amperometry and sinusoidal voltammetry

A microchip electrophoresis system with integrated electrochemical detection is described in this work. The hybrid device utilizes poly(dimethylsiloxane) as the electrophoresis channel substrate and a planar gold electrode lithographically fabricated onto a glass slide for electrochemical detection. The system is characterized by the separation and detection of various neurotransmitters. The gold working electrode is placed just inside the separation channel without adverse effects on the detection sensitivity, due to the electrical decoupling of the detection and electrophoresis systems. The close proximity of the working electrode to the exit of the separation channel results in symmetric peak shapes and efficient separations (50,000-100,000 plates/m). A direct comparison between the frequency-based electrochemical technique, sinusoidal voltammetry, and the more commonly used constant potential (DC) amperometry is made. Sinusoidal voltammetry is found to be roughly an order of magnitude more sensitive than DC amperometry, with calculated mass detection limits (S/N = 3) of 12 amol and 15 amol for dopamine and isoproterenol, respectively.     

New developments in planar array infrared spectroscopy

A planar array infrared (PA-IR) spectrograph offers several advantages over other infrared approaches, including high acquisition rate and sensitivity. However, it suffers from some important drawbacks, such as a limited spectral range and a significant curvature of the recorded spectral images, which still need to be addressed. In this article, we present new developments in PA-IR spectroscopy that overcome these drawbacks. First, a data processing method for the correction of the curvature observed in the spectral images has been developed and refined. In addition, a dual-beam instrument that allows the simultaneous recording of two independent spectral images has been developed. These two improvements have been combined to demonstrate the real-time background correction capability of PA-IR instruments. Finally, the accessible spectral range of the PA-IR spectrograph has been extended to cover simultaneously the methylene stretching (3200-2800 cm(-1)) and the finger-print (2000-1000 cm(-1)) spectral regions.     

Affinity capture and detection of immunoglobulin E in human serum using an aptamer-modified surface in matrix-assisted laser desorption/ionization mass spectrometry

Capture and detection of immunoglobulin E (IgE) in simple solution and in human serum using an aptamer-modified probe surface for affinity matrix-assisted laser desorption/ionization mass spectroscopy detection is reported. Detectable signals were obtained for 1 amol of IgE applied either in a single, 1microL application of 1 pM IgE or after 10 successive, 1-microL applications of 100 fM IgE. In both cases, the surface was rinsed after each application of IgE to remove sample concomitants including salts and free or nonspecifically associated proteins. Detection of native IgE, which is the least abundant of the serum immunoglobulins and occurs at subnanomolar levels, in human serum was demonstrated and interference from the high-abundance immunoglobulins and albumin was investigated. The aptamer-modified surface showed high selectivity toward immunoglobulins in serum, with no significant interference from serum albumin. Addition of IgE to the serum suppressed the signals from the other immunoglobulins, confirming the expected selectivity of the aptamer surface toward IgE. Dilution of the serum increased the selectivity toward IgE; the protein was detected without interference in a 10,000-fold dilution of the serum, which is consistent with detection of IgE at amol (pM) levels in standard solutions.     

High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.     

High-performance immunoaffinity chromatography and chemiluminescent detection in the automation of a parathyroid hormone sandwich immunoassay

An automated sandwich immunoassay was developed based on high-performance immunoaffinity chromatography and chemiluminescent detection, using the determination of parathyroid hormone (PTH) in plasma as a model system. In this method, injections of plasma and acridinium ester-labeled anti-(1-34 PTH) antibodies were made onto a column containing immobilized anti-(44-68 PTH) antibodies. Upon elution, PTH and its associated labeled antibody were combined with an alkaline peroxide postcolumn reagent, and the resulting light production was measured. Factors considered in optimizing this system included the column's dissociation properties, the rate of light production in the postcolumn reactor, and the use of sequential vs simultaneous injection of sample and labeled antibody. The final system developed required 6 min per plasma injection, following a 1-h incubation of sample with labeled antibody. The response was linear over 2-3 orders of magnitude and the lower limit of detection for a 66-microL plasma sample was only 16 amol, or 2.4 x 10(-13) M. Overall, this method had a precision and response similar to those of manual PTH methods but required 24-fold less time to perform. By using different immobilized and labeled antibodies, this method could easily be adapted for use with other analytes.     

Capillary electrophoresis and fluorescence anisotropy for quantitative analysis of peptide-protein interactions using JAK2 and SH2-Bbeta as a model system

Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide-protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-Bbeta (SH2-Bbeta (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the K(d) = 82 +/- 7 nM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2-Bbeta (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing K(d) (101 +/- 12 nM in good agreement with FA measurements) and dissociation rate (k(off) = 0.95 +/- 0.02 s(-)(1) corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining K(d) or k(off); however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics.     

Online Magnetic Flux Leakage Detection System for Sucker Rod Defects Based on LabVIEW Programming

Aiming at the detection of the sucker rod defects, a real-time detection system is designed using the non-destructive testing technology of magnetic flux leakage (MFL). An MFL measurement system consists of many parts, and this study focuses on the signal acquisition and processing system. First of all, this paper introduces the hardware part of the acquisition system in detail, including the selection of the Hall-effect sensor, the design of the signal conditioning circuit, and the working process of the single chip computer (SCM) control serial port. Based on LabVIEW, a graphical programming software, the software part of the acquisition system is written, including serial port parameter configuration, detection signal recognition, original signal filtering, real-time display, data storage and playback. Finally, an experimental platform for the MFL detection is set up, and the MFL measurement is carried out on the transverse and longitudinal defects of the sucker rod surface. The experimental result shows that the designed acquisition and processing system has good detection performance, simple design and high flexibility.     

Analysis of nucleic acids by capillary ion-pair reversed-phase HPLC coupled to negative-ion electrospray ionization mass spectrometry

Ion-pair reversed-phase high-performance liquid chromatography was successfully coupled to negative-ion electrospray ionization mass spectrometry by using 60 × 0.20 mm i.d. capillary columns packed with 2.3-μm micropellicular, octadecylated poly(styrene/divinylbenzene) particles as stationary phase and gradients of acetonitrile in 50 mM aqueous triethylammonium bicarbonate as mobile phase. Systematic variation of the eluent composition, such as concentration of ion-pair reagent, anion in the ion-pair reagent, solution pH, and acetonitrile concentration led to the conclusion that most parameters have opposite effects on chromatographic and mass spectrometric performances. The use of acetonitrile as sheath liquid enabled the rapid and highly efficient separation and detection of phosphorylated and nonphosphorylated oligonucleotides ranging in size from 8 to 40 nucleotides. High-quality full-scan mass spectra showing little cation adduction were acquired from which the molecular masses of the separated oligonucleotides were calculated with an accuracy of 0.011%. With calibration curves being linear over at least 2 orders of magnitude, the lower limits of detection for a oligodeoxythymidine 16-mer were 104 fmol with full scan and 710 amol with selected-ion-monitoring data acquisition. The potential of ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was demonstrated for mixed-sequence oligomers by the characterization of a reaction mixture from solid-phase synthesis of a 40-mer oligonucleotide.     

Detection of peptides by precolumn derivatization with biuret reagent and preconcentration on capillary liquid chromatography columns with electrochemical detection

The separation and detection of biuret complexes of neuropeptides by capillary liquid chromatography with electrochemical detection was explored. Capillaries of 25-micron inner diameter packed with base-resistant, polymer-based reversed-phase particles were used for separation, and C-fiber electrodes were used for detection. Detection at the C-fiber electrode was found to have some differences in relative sensitivity for peptides compared to glassy carbon electrodes used previously. On-column preconcentration of preformed complexes allowed up to 1-microL samples to be injected with minimal band broadening resulting in a 100-fold improvement in concentration detection limit with no effect on mass detection limit. Concentration detection limits ranged from 5 to 59 pM, depending upon the peptide, corresponding to 5-59 amol injected. The low concentration detection limit was possible because of minimal baseline disturbances, minimal formation of unwanted products, and high efficiency of complex formation associated with biuret derivatization. The method was applied to determination of vasopressin and bradykinin in dialysates collected with 5-min sampling frequency from the rat supraoptic nucleus.     

Batch-type chemiluminescence detection cell for sensitization and simplification of capillary electrophoresis

A simple and convenient chemiluminescence detection cell was designed for capillary electrophoresis. The detection cell easily combined with capillary electrophoresis equipment. Luminol chemiluminescence was adapted for use with the detection cell. Detailed analysis and testing of the system revealed that luminol could be determined over a range of 2.5 x 10(-10)-6.5 x 10(-7) M (correlation coefficient, 0.999), with a detection limit (S/N = 3) of 2.5 x 10(-10) M (7 amol). Furthermore, each component in a mixture of glycine, glycylglycine, and glycylglycylgycine, which were labeled with isoluminol isothiocyanate, was baseline separated and sensitively detected. Moreover, the stacking procedure was applied to postcolumn detection in capillary electrophoresis. When acetonitrile stacking was used under certain conditions in the present system, chemiluminescence intensities of luminol and labeled compounds were about 1 order of magnitude higher than those obtained without stacking. The detection limit for luminol was 1.5 x 10(-11) M (S/N = 3), representing the highest sensitivity of luminol yet reported. Finally, the effect of p-iodophenol as an enhancer of luminol chemiluminescence was examined under weak alkaline conditions. The chemiluminescence intensity of luminol was approximately 2 orders of magnitude higher than that in the unenhanced reaction. A preliminary immunoassay using horseradish peroxidase-labeled anti-mouse IgG was also developed.     

Determination of naphthalene-2,3-dicarboxaldehyde-labeled amino acids by open tubular liquid chromatography with electrochemical detection

Naphthalene-2,3-dicarboxaldehyde (NDA) has been investigated as a new derivatizing reagent for the electrochemical detection of tagged amino acids. Gradient elution allowed for the separation of 18 NDA-derivatized amino acids on an open tubular liquid chromatography column in less than 50 min. Gradient elution and electrochemical detection were found to be compatible. A detection limit of 36 amol was obtained for the asparagine-NDA derivative. The usefulness of this technique for quantitation was demonstrated by the analysis of the NDA-tagged hydrolysis products from bovine chymotrypsinogen.     

Detection of G proteins by affinity probe capillary electrophoresis using a fluorescently labeled GTP analogue

An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.     

频域光存储和全息术

本文通过分析光谱烧孔的全息探测法,提出了全息频域光存储系统,并分析了系统的特性指标。计算机模拟分析结果表明,全息频域光存储技术是一种大容量并行光存储新技术。