Determination of various hemoglobin species with thermal-lens spectrometry

Desoxyhemoglobin, oxyhemoglobin, carboxyhemoglobin, methemoglobin, cyanohemoglobin, and hemichrome are determined using thermal-lens spectrometry, with the detection limits at the level of 10^?8 M/l (2–5 mcg/l depending on hemoglobin species). Signal behavior and detectability thresholds are in good agreement with theoretical modeling based on the approach proposed earlier by the authors to describe thermal-lens signal generation in complex (inhomogeneous) systems. The thermooptical response for all studied hemoglobin species depends on the power of laser radiation within the range of 1–50 mW (532, 514.5, and 488 nm). Under the conditions of a thermal-lens experiment, the total temperature growth (0.0001 K) due to heating of the studied solution as the radiation of the inducing laser is absorbed by hemoglobin is estimated. Due to the interference with oxyhemoglobin the error in determining desoxyhemoglobin using thermal-lens spectrometry (for a maximum radiation power of 532 nm, 210 mW) does not exceed 3% in the case when the ratio of the species is 10: 1. In the opposite case (determination of oxyhemoglobin in the presence of desoxyhemoglobin), the error does not exceed 5%, the ratio of test and interfering species being the same.     

Determination of diacetyl with spectrophotometry and thermal-lens spectrometry

The conditions for the spectrophotometric and thermal-lens determination of diacetyl with creatine and 2-naphthol are proposed. The obtained value of the detection limit for spectrophotometry (at 527 nm), which amounts to 10 ng/mL, is fivefold lower than the existing values of the spectrophotometric determination of diacetyl. The conditions for the thermal-lens determination of diacetyl (λ = 514.5 nm, strength of the inducing radiation: 40 mW) based on the unmodified procedure of spectrophotometric determination were proposed. Along with a fivefold (down to 2 ng/mL) decrease in the detection limit, which is comparable with that for the determination of diacetyl by means of gas chromatography with mass spectrometric detection (detection limit of 0.7 ng/mL), thermal-lens determination is characterized by the enhancement of other performance parameters of the determination. It was shown that, contrary to the case when gas chromatography is used, ethanol does not interfere with both the spectrophotometric and the thermal-lens determination of acetyl.     

Determination of copper with neocuproine by thermal-lens spectrometry

Conditions for the spectrophotometric determination of copper with 2,9-dimethyl-l,10-phenan-throline (neocuproine) in the presence of ascorbic acid in a water-ethanol solution (9 : 1) at pH 4.5–5.0 have been found. The detection limit is 3 x 10^-6 M. The concentration range is from 4.4 x 10^-6 to 3 x 10^-4 M. Conditions for the determination of copper(I) with neocuproine by thermal lens spectrometry have been proposed. The detection limit is 4 x 10^-7 M. The concentration range is from 7 x 10^-7 to 6 x 10^-5 M. Iron(II) at concentrations as high asn x 10^-4 M does not interfere with the determination of copper. Changes in the conditions for the photometric reaction associated with passing from spectrophotometric measurements to thermal lensing are discussed.     

Integrated liquid chromatography-heated nebulizer microchip for mass spectrometry

A new integrated microchip for liquid chromatography-mass spectrometry (LC-MS) is presented. The chip is made from bonded silicon and glass wafers with structures for a packed LC column channel, a micropillar frit, a channel for optional optical detection, and a heated vaporizer section etched in silicon and platinum heater elements on the glass cover. LC eluent is vaporized and mixed with nebulizer gas in the vaporizer section and the vapor is sprayed out from the chip. Nonpolar and polar analytes can be efficiently ionized in the gas phase by atmospheric pressure photoionization (APPI) as demonstrated with polycyclic aromatic hydrocarbons (PAHs) and selective androgen receptor modulators (SARMs). This is not achievable with present LC-MS chips, since they are based on electrospray ionization, which is not able to ionize nonpolar compounds efficiently. The preliminary quantitative performance of the new chip was evaluated in terms of limit of detection (down to 5 ng mL⁻¹), linearity (r > 0.999), and repeatability of signal response (RSD = 2.6-4.0%) and retention time (RSD = 0.3-0.5%) using APPI for ionization and PAHs as standard compounds. Determination of fluorescent compounds is demonstrated by using laser-induced fluorescence (LIF) for detection in the optical detection channel before the vaporizer section.     

Investigation of ionene adsorption on quartz surfaces by thermal-lens spectrometry

Thermal-lens spectrometry was used for the investigation of the adsorption of ionene to quartz surfaces. The thermooptical analysis of the surface makes it possible to distinguish the modified surface from a clean quartz surface and to provide sensitive direct concentration measurements of the light absorbing co-adsorbed substance. The co-adsorption of chromate ions and 2,10-ionene from aqueous solutions to quartz surfaces was investigated and the desorption procedure proposed.     

Robust and simple interface for microchip electrophoresis-mass spectrometry

A robust and simple interface for microchip electrophoresis-mass spectrometry (MCE-MS) was developed using a spray nozzle connected to the exit of the separation channel of the microchip. The spray nozzle was attached to the microchip using a polyether ether ketone screw without adhesive, thus allowing easy replaced. Sample injection and electrophoretic separation was performed by control of the voltage only. The analysis of a few basic drugs was performed using the optimized MCE-MS system. The separation was improved by using a high-viscosity separation buffer and a spray nozzle with a small bore size. This system was also applied to the separation of peptides and protein-trypsin digests. Sample adsorption was minimized by adding acetonitrile to the separation buffer when using a quartz microchip.     

Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (microchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (microchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by microchip-CE/MS on quadrupole (Q) and time-of-flight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by microchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this microchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by microchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, microchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.     

A novel approach to the regioselective synthesis of a disulfide-linked heterodimeric bicyclic peptide mimetic of brain-derived neurotrophic factor

We describe a novel acetamidomethyl to S-pyridinyl exchange that is used for the synthesis of a multi-disulfide-linked and constrained heterodimeric bicyclic peptide mimetic of brain-derived neurotrophic factor (BDNF). This simple and effective method should be readily transferable to the synthesis of similar disulfide-linked heterodimeric peptides, as well as being of general utility for the synthesis of peptides bearing multiple cystine frameworks.     

Top‐down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

The original article to which this Erratum refers was published in Rapid Commun. Mass Spectrom. 2006; 20 : 1932–1938.     

Electroactive intercalators for DNA analysis on microchip electrophoresis

Miniaturized analytical systems, especially microchip CE (MCE), are becoming a promising tool for analytical purposes including DNA analysis. These microdevices require a sensitive and miniaturizable detection system such as electrochemical detection (ED). Several electroactive DNA intercalators, including the organic dye methylene blue (MB), anthraquinone derivatives, and the metal complexes Fe(phen)3 2+ and Ru(phen)3 2+, have been tested for using in combination with thermoplastic olefin polymer of amorphous structure (Topas) CE-microchips and ED. Two end-channel approaches for integration of gold wire electrodes in CE-ED microchip were used. A 250 microm diameter gold wire was manually aligned at the outlet of the separation channel. A new approach based on a guide channel for integration of 100 and 50 microm diameter gold wire has been also developed in order to reduce the background current and the baseline noise level. Modification of gold wire electrodes has been also tested to improve the detector performance. Application of MCE-ED for ssDNA detection has been studied and demonstrated for the first time using the electroactive dye MB. Electrostatic interaction between cationic MB and anionic ssDNA was used for monitoring the DNA on microchips. Thus, reproducible calibration curves for ssDNA were obtained. This study advances the feasibility of direct DNA analysis using CE-microchip with ED.     

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%.     

Frontal analysis on a microchip

Conventional microchip applications involving capillary electrophoresis (CE) typically inject a sample along one channel and use an intersection of two channels to define the sample plug--the portion of sample to be analysed along a second channel. In contrast to this method of zone separation, frontal analysis proceeds by injecting sample continuously into a single channel or column. Frontal analysis is more common in macroscopic procedures but there are benefits in sensitivity and device density to its application to electrophoresis on microchips. This work compares conventional microchip zone analysis with frontal analysis in the separation of PCR products. Although we detect on the order of 5000 fluorophores with a compact instrument using the zone separation CE method, we found a several-fold increase in the effective signal-to-noise ratio by using a frontal analysis method. By removing the need for additional channels and reservoirs the frontal method would allow device densities to be significantly increased, potentially improving the cost-effectiveness of microchip analyses in applications such as medical diagnostics.     

Recent advances in microchip electrophoresis for amino acid analysis

With the maturation of microfluidic technologies, microchip electrophoresis has been widely employed for amino acid analysis owing to its advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. In this article, we review the recent progress in amino acid analysis using microchip electrophoresis during the period from 2007 to 2012. Innovations in microchip materials, surface modification, sample introduction, microchip electrophoresis, and detection methods are documented, as well as nascent applications of amino acid analysis in single-cell analysis, microdialysis sampling, food analysis, and extraterrestrial exploration. Without doubt, more applications of microchip electrophoresis in amino acid analysis may be expected soon.     

Desktop near-field thermal-lens microscope for thermo-optical detection in microfluidics

A new compact near-field desktop-sized diode laser thermal-lens microscope for analysis in microfluidics was proposed. A novel beam-alignment and detection systems provided high signal stability and, along with reduced number of optical elements rendered the instrument portable. The detection of nonfluorescent model species (Fe(II)-bathophenanthroline chelate) in water showed good linearity in the range of 5 × 10(-9) to 1 × 10(-4) M, and the limit of detection was 3.5 × 10(-9) M, which corresponded to 3.5 × 10(-7) absorbance units and provided a 20-fold enhancement in sensitivity compared with existing schematic.     

High-throughput DNA analysis by microchip electrophoresis

DNA analysis plays a great role in genetic and medical research, and clinical diagnosis of inherited diseases and particular cancers. Development of new methods for high throughput DNA analysis is necessitated with incoming of post human genome era. A new powerful analytical technology, called microchip capillary electrophoresis (MCE), can be integrated with some experimental units and is characterized by high-speed, small sample and reagent requirements and high-throughput. This new technology, which has been applied successfully to the separation of DNA fragments, analysis of polymerase chain reaction (PCR) products, DNA sequencing, and mutation detection, for example, will become an attractive alternative to conventional methods such as slab gel electrophoresis, Southern blotting and Northern blotting for DNA analysis. This review is focused on some basic issues about DNA analysis by MCE, such as fabrication methods for microchips, detection system and separation schemes, and several key applications are summarized.     

Automation for continuous analysis on microchip electrophoresis using flow-through sampling

Automation of electrophoretic microchips for sequential analysis of different samples is demonstrated. This system used an autosampler, which was on-line connected to the microchip and the whole process including sample loading and injection, analysis and data acquisition as well as washing were all automated. Rhodamin B at different concentrations was first loaded into a hydrodynamic flow stream by an autosampler, delivered to the microchip, and then sequentially injected into the electrophoretic microchannel for analysis and detection. Automation was achieved by running two independent programs, one for sample loading by an autosampler and the other one for electrophoretic injection by voltage switching, on the same computer. Using this sampling chip, each loaded volume (0.2-1 microL) can be injected for dozens of electrophoretic analyses (1-10 nL for each injection). The variances caused by the external connections, which did not affect the electrophoretic analysis but would cause band broadening of the loaded sample in the hydrodynamic flow stream, were theoretically deduced. Results indicate that the dead volume (approximately 300 nL) due to the connection fitting on the chip could lead to dilution of the loaded sample by a factor of one when 0.2 microL of sample was loaded. Such a design allows sequential analysis of a series of samples while the running buffer is continuously pumped into the connection capillary as well as microchannels for washing between two loaded samples to minimize cross contamination without human intervention. Using this sampling chip, the required sample amount and handling time can be greatly reduced compared to the manual method.     

Structure-transport analysis for particulate packings in trapezoidal microchip separation channels

This article investigates the efficiency of particulate beds confined in quadrilateral microchannels by analyzing the three-dimensional fluid flow velocity field and accompanying hydrodynamic dispersion with quantitative numerical simulation methods. Random-close packings of uniform, solid (impermeable), spherical particles of diameter d(p) were generated by a modified Jodrey-Tory algorithm in eighteen different conduits with quadratic, rectangular, or trapezoidal cross-section at an average bed porosity (interparticle void fraction) of epsilon = 0.48. Velocity fields were calculated by the lattice Boltzmann method, and axial hydrodynamic dispersion of an inert tracer was simulated at Péclet numbers Pe = u(av)d(p)/D(m) (where u(av) is the average fluid flow velocity through a packing and D(m) the bulk molecular diffusion coefficient) from Pe = 5 to Pe = 30 by a Lagrangian particle-tracking method. All conduits had a cross-sectional area of 100d(p)(2) and a length of 1200d(p), translating to around 10(5) particles per packing. We present lateral porosity distribution functions and analyze fluid flow profiles and velocity distribution functions with respect to the base angle and the aspect ratio of the lateral dimensions of the different conduits. We demonstrate significant differences between the top and bottom parts of trapezoidal packings in their lateral porosity and velocity distribution functions, and show that these differences increase with decreasing base angle and increasing base-aspect ratio of a trapezoidal conduit, i.e., with increasing deviation from regular rectangular geometry. Efficiencies are investigated in terms of the axial hydrodynamic dispersion coefficients as a function of the base angle and base-aspect ratio of the conduits. The presented data support the conclusion that the efficiency of particulate beds in trapezoidal microchannels strongly depends on the lateral dimensions of the conduit and that cross-sectional designs based on large side-aspect-ratio rectangles with limited deviations from orthogonality are favorable.     

A polymeric microchip with integrated tips and in situ polymerized monolith for electrospray mass spectrometry

We describe the integration of a cyclo-olefin polymer based microchip with a sheathless capillary tip for electrospray ionization-mass spectrometry (ESI-MS). The microchip was fabricated by hot embossing and thermal bonding. Its design includes a side channel for adjusting the composition of the electrospray solution so that analytes in 100% water can be analyzed. The fused silica capillaries, used for sample introduction, and the electrospray tips for MS coupling were directly inserted into the microchannel before thermal bonding of the device. A microfabricated on-chip gold microelectrode was used to apply the electrospray voltage. Annealing the device after thermal bonding increased the pressure resistance of the microchip. The cross section of the microchannel was imaged by scanning electron microscopy to estimate the effects of the annealing step. The relationship between the applied electrospray voltages and MS signal was measured at different flow rates by coupling the device to an ion trap mass spectrometer. The performance of the microchip was evaluated by MS analysis of imipramine in ammonium acetate buffer solution by direct infusion. An alkylacrylate based monolith polymer bed for on-chip sample pretreatment and separation was polymerized in the microchannel and tested for ESI-MS applications.     

A simplified method for the determination of total homocysteine in plasma by electrospray tandem mass spectrometry

Hyperhomocysteinemia is a risk factor for different diseases. Several methods have been developed to analyze homocysteine and the immunometric ones, although expensive, they are in widespread use. A rapid LC‐MS/MS method for homocysteine assay has been developed for the application of large clinical chemistry routines. Selected reaction monitoring was performed through the transitions m/z 136.0→90.1 for homocysteine and m/z 140.0→94.0 for the internal standard. ESI was used to generate [H+] adduct ions. Chromatographic isocratic separation was achieved using a strong cation exchange column. The mobile phase was methanol/water (20:80 v/v, containing 0.1% formic acid and 1.5 mmol/L ammonium formate in the water phase) at a flow rate of 0.250 mL/min (35°C). Samples treatment consisted in the reduction with DTT and deproteinization with methanol. Recovery, linearity, LOD, LOQ and total imprecision were evaluated to validate the method. Homocysteine values on 100 serum samples were compared with those obtained by HPLC and immunometric methods. The method is robust, selective and precise in the whole range of values studied. Moreover, low reagent cost and easiness of sample treatment make this method useful, not only for research, but also for routine work.