Optical techniques for identifying animal and plant pathogens

Abstract

The contemporary revolution in biochemistry, bio-physics, and medicine is leading to an understanding of how biological entities operate in varied environments. One consequence is the increased understanding of antibodies, fluorescence markers, special substrates, and uses of the polymerase chain reaction process with biological markers. In particular, these efforts are leading to pathogen detection systems that are lower in cost, more biochemically specific, more accurate, faster, smaller, less demanding of infrastructure, and more accessible to more people than ever before. Most of these systems use optical techniques. Examples of miniature, portable detection systems based upon nucleotide amplification and on fluorescence sorting, concentration, and detection are discussed. These are applicable to the detection of pathogens and related toxins that appear in medical, agricultural, and national security situations confronting us today.     

Computer simulation of fast electromigration lifetime determination techniques

The electromigration lifetime and the activation energy of the process are often determined by highly accelerated or fast methods. Some of these common methods have been simulated using a crack model of the defect. The TRACE, BEM and WIJET methods are considered and suggestions are made about the optimum test strategy.     

Separation of serotonin from catechols by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis with electrochemical detection is demonstrated with columns having only 9-microns inner diameter. Amperometric detection limits of 0.7 amol are reported for serotonin. The difficult problem of resolving serotonin and dopamine--two neurotransmitters of interest having similar electrophoretic mobilities--is addressed by chemical means to improve selectivity. These include buffer modification with 2-propanol and a system employing borate complexation of the catechol in combination with sodium dodecyl sulfate micelles.     

Separation of fines by flotation techniques

A large amount of valuable minerals is discarded today as fines and ultrafines because of inadequate technology to process them economically. Treatment of fine particles presents a difficult problem in the chemical industry and raw materials processing, and its solution is required both in production and effluent treatment. This article reviews various suitable flotation techniques—such as precipitate flotation, flocculation-flotation, column flotation, dissolved-air flotation, and electrolytic flotation and presents different laboratory experiments with fines (mostly at the subsieve size range) as examples. Also included is the conventional froth flotation and particularly the effect of bubbles size on performance. Although froth flotation is a commonly applied selective separation process in mineral processing, it becomes inefficient for beneficiating fines. In the closing overview of the process, several ideas for future research are suggested.     

Microhardness of ceramics produced from different alumina nanopowders by different techniques

Fine-grained ceramics (with a grain size on the order of a micron) have been produced by the spark plasma sintering (SPS) of various alumina nanopowders. We have compared the microhardness of ceramic samples prepared from 11 alumina nanopowders and that of composites based on such powders. The ceramics have been prepared by both SPS and a conventional technique (sequential pressing and sintering). We examine the effect of the phase composition and average particle size of the starting nanopowder on the microhardness of the ceramics.     

Separation of acidic and basic proteins by nanoparticle-filled capillary electrophoresis

We present the first example of the analysis of acidic and basic proteins by nanoparticle-filled capillary electrophoresis. Compared to the didodecyldimethylammonium bromide (DDAB)-coated capillary, the DDAB-capped gold nanoparticles (AuNPs) as pseudostationary phase were found to form more stable coating on the capillary wall, thus leading to greater separation efficiency and high reproducibility. In addition to their advantages for protein separation, DDAB-capped AuNPs can generate high reversed electroosmotic flow, which is 75% greater than DDAB at pH 3.5. To allow strong interactions with proteins, the AuNPs were modified with poly(ethylene oxide) via noncovalent bonding to form gold nanoparticles/polymer composites (AuNPPs). Using a capillary dynamically coated with DDAB-capped AuNPs and filled with AuNPPs under acidic conditions (10 mM phosphate, pH 3.5), we have demonstrated the separation of acidic and basic proteins with peak efficiencies ranging from 71 000 to 1 007 000 plates/m and relative standard deviations of migration time less than 0.6%. Additionally, the proposed method has been applied to the analyses of biological samples, including saliva, red blood cells, and plasma. With simplicity, high resolving power, and high reproducibility, the proposed method has shown great potential for proteomics applications and clinical diagnosis.     

Separation of long double-stranded DNA by nanoparticle-filled capillary electrophoresis

We present the first example of the analysis of long double-stranded (ds) DNA molecules by nanoparticle-filled capillary electrophoresis (NFCE). To avoid aggregation of the gold nanoparticles (GNPs) and to allow strong interactions with the DNA molecules, the gold nanoparticles were modified with poly(ethylene oxide) (PEO) via noncovalent bonding to form gold nanoparticle/polymer composites (GNPPs). The neutral GNPPs are heavy (approximately 2.0 x 10(8) g/mol for the 32-nm GNP) and thus slow the DNA molecules that they encounter during the electrophoretic process. Compared to linear polymer solutions, such as hydroxyethyl cellulose and PEO, the GNPPs provide greater efficiency and require significantly shorter times to separate long dsDNA. The separation of lambda-DNA (0.12-23.1 kbp) by NFCE at -250 V/cm was accomplished in 3 min. The ability to separate high molecular weight DNA markers (8.27-48.5 kbp) with plate numbers greater than 10(6) suggests that this novel method may hold great promise for the analysis of long-stranded DNA molecules such as chromosomes. Moreover, this method is simple and affordable when compared to those that use micro- and nanofabricated devices for separating long DNA molecules.     

Separation of nonionic surfactants by capillary electrophoresis and high-performance liquid chromatography

Fatty alcohol ethoxylates (FAEs) are applied in commercial formulations (laundry detergents) as complex mixtures of alkyl and ethoxylate homologues. Therefore, efficient analytical methods are required for product control. Capillary electrophoresis, a modern analytical separation technique, was used to separate FAEs in technical products and household formulations after derivatization with phthalic anhydride. The well-established high-performance liquid chromatography was used as reference and supplementary method. UV detection after derivatization with phenyl isocyanate or light scattering detection has been carried out. Sample components have been identified by standard addition or by comparison to known products. The peak pattern can be considered as a "fingerprint" of the product and is characteristic for a defined composition.     

Separation and detection of intact noncovalent protein complexes from mixtures by on-line capillary isoelectric focusing-mass spectrometry

Separation and mass spectrometric analysis of intact noncovalent protein-protein complexes from mixtures is described. Protein complexes were separated using isoelectric focusing in a capillary under native conditions. During the mobilization, molecular masses of the intact complexes were measured on-line (as they emerged from the capillary) using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. An FTICR "in-trap" ion cleanup procedure was necessary for some complexes to reduce high levels of adduction and to obtain accurate molecular mass measurements. Optimization of the conditions for analysis of different intact complexes is discussed. We have shown that either the intact noncovalent complexes or their constituent protein subunits can be detected by variation of sheath liquid (i.e., NH4OAc vs HOAc) added at the electrospray-mass spectrometer interface. Thus, two successive experiments permit a fast and efficient characterization of intact complex stoichiometry, the individual complex subunits and the possible presence of metal or other adducted species.     

Dependence of electromigration caused by different mechanisms on current densities in VLSI interconnects

In this paper, we have studied electromigration (EM) behavior under different current densities experimentally and theoretically. Our experimental results have shown that the dependence of median-time-to-failure on current density is not the same in different regimes of current densities. Both the theoretical analysis and observation of SEM showed that mechanisms causing EM failure in different regimes are different. In the low current density regime EM failure is caused by microstructural changes, while in the high current density regime it is microstructural changes caused by a temperature gradient. We have developed a model to describe the dependence. The calculated results are consistent with experiments.     

Separation of explosives using capillary electrochromatography

The identification of explosives and their degradation products is important in forensic and environmental applications. Complete separation of these structurally similar compounds using reversed-phase liquid chromatography has proven to be a challenge. Here we present a demonstration of the use of capillary electrochromatography on the separation of a series of 14 nitroaromatic and nitramine explosive compounds. A separation with baseline resolution is achieved for all of the compounds in under 7 min, featuring efficiencies of over 500?000 theoretical plates/m. Using more aggressive running conditions, 13 of the 14 compounds are separated in under 2 min.     

Separation of chiral biodegradation intermediates of linear alkylbenzenesulfonates by capillary electrophoresis

High-performance capillary electrophoresis (HPCE) with α-cyclodextrin as the chiral selector was applied to separate the enantiomers of p-sulfophenyl-2-butyrate (SP2B) and p-sulfophenyl-3-butyrate (SP3B), which occur as biodegradation intermediates of linear alkylbenzenesulfonates (LAS), the widely used anionic surfactants. With this analytical method, we studied the transformation of both SP3B enantiomers in a laboratory batch incubation with activated sewage sludge of a municipal wastewater treatment plant. (S)-(+)-SP3B and (R)-(-)-SP3B could be detected in mechanically treated sewage effluent. After enrichment on graphitized carbon black (Carbopack B), the extracts were analyzed by HPLC with UV diode array and fluorescence detection as well as by HPCE with UV diode array detection. Quantification of SP3B in a 24-h composite sample of primary sewage effluent yielded 34 μg/L (limit of detection, 0.1 μg/L) of the racemic mixture determined by HPLC and 18 μg/L of each enantiomer measured by HPCE (limit of detection, 1 μg/L).     

Separation and quantitation of short-chain coenzyme A's in biological samples by capillary electrophoresis

Because of the importance of coenzyme A's (CoA's or CoASH) in many metabolic processes and the biosynthesis of some carbohydrates and lipids, many methods have been developed to separate and determine their levels in various tissues for metabolism studies, including enzymatic assays, paper chromatography, and high-performance liquid chromatography (HPLC). However, inadequate separation of coexisting CoA's in biological samples was often encountered due to the similarity of their structures. In this paper, we demonstrated for the first time the separation and quantitation of 12 different CoA's by using capillary electrophoresis with UV detection at 254 nm. All 12 CoA's (CoASH, HMG CoA, methylmalonyl CoA, succinyl CoA, methylcrotonyl CoA, isobutyryl CoA, oxidized CoA, acetyl CoA, crotonoyl CoA, n-propzoyl CoA, acetoacetyl CoA, malonyl CoA) were completely separated at -30 kV in a 100 mM NaH2PO4 running buffer containing 0.1% beta-cyclodextrin at pH 6.0. The total separation time was less than 30 min. The signal response was linear over 2 orders of magnitudes (from 1 to 100 nmol), and the detection limits were in the picomole range. The effects of pH, buffer concentration, additives, and operation voltages on sensitivity and resolution were also discussed. This technique, described here, is much more sensitive, faster, and simpler than the published HPLC methods and can potentially be used for mechanistic study in biological systems involving CoA metabolism.     

Morphological evolution of edge-hillocks on single-crystal films having anisotropic drift-diffusion under the capillary and electromigration forces

The morphological evolution of hillocks at the unpassivated sidewalls of single-crystal metallic thin film interconnects is investigated via computer simulations using the free-moving boundary value problem. The effect of drift-diffusion anisotropy on the development of surface topographical scenarios is fully explored under the action of electromigration and capillary forces, utilizing numerous combinations of the surface texture, the drift-diffusion anisotropy and the direction of the applied electric field. The simulation studies yield analytical relationships for the velocity of the surface solitary waves and the drift velocity of electromigration-induced internal voids as a function of the applied current densities, which contain intrinsic and structural properties of the single-crystal thin films. The threshold value of the applied current density, above which electromigration-induced internal voids can be formed and may cause the catastrophic failure of interconnects by breaching, also appears explicitly in this relationship.     

Separation of single-walled carbon nanotubes by use of ionic liquid-aided capillary electrophoresis

This paper presents a simple, highly efficient method for analyzing single-walled carbon nanotube (SWNT) bundles based on (1) ultrasound-assisted solubilization/dispersion of SWNTs in the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate, (2) encapsulation of the nanotubes in sodium dodecyl sulfate micelles, and (3) analysis by capillary electrophoresis. The process by which SWNTs disperse in the ionic liquid was studied by Raman spectroscopy. No degradation of SWNTs was observed under mild sonication conditions. The shape and position changes observed in the Raman spectral bands for the nanotubes are ascribed to debundling and interaction with the ionic liquid. Separation of solubilized SWNTs was accomplished by using a 50 mM formic acid solution at pH 2.0 as background electrolyte and a potential of -10 kV. Under these conditions, separation was completed within only 4 min. Eighteen peaks for SWNTs were identified in the analysis of commercial SWNT bundles. The two types of bundles studied exhibited distinct, highly characteristic electrophoretic profiles which could be used to control SWNTs purity.     

Separation of tissue proteins of human lung carcinomas by partial-filling capillary electrophoresis

Tissue proteins from human squamous cell lung carcinomas (SQCLC) and small cell lung carcinomas (SCLC) were separated in 0.01% hydroxypylmethyl cellulose (HPMC) linear polymer sieving solutions in the inlet portion of the capillary and next to the outlet of the capillary, followed by capillary zone electrophoresis (CZE) in 40 mM phosphate buffer, pH 2.5. A proper HPMC concentration could cause a molecular sieving effect through the formation of an entangled polymer network. The migration time of the analyte in this matrix depended on the size and electrophoretic mobility of the analyte, the mesh size, and the electric field strength. In the CZE separation, the electroosmotic flow and the charge-to-size ratio of the analyte were important parameters. HPMC concentration and zone length were examined to optimize the separation. Applying this partial-filling technique to the separation of water-soluble proteins from human lung tissues, we found a greatly improved resolution and increased peak intensity. The capillary electrophoresis patterns of normal, SQCLC, and SCLC were obtained and compared for their molecular classifications.     

Carbonate loss from two magnesium-substituted carbonated apatites prepared by different synthesis techniques

This study was aimed at the investigation of the thermal stability of Mg-substituted carbonated apatites over the wide temperature range. Two different apatites were studied, which were prepared by either precipitation from aqueous solution or by solid–liquid interaction. The following methods were employed: FTIR spectroscopy of the condensed gas phase to evaluate the CO and CO₂ release with increasing temperature, FTIR of the solid residue after heating, XRD analysis, thermogravimetry and scanning electron microscopy. Decomposition behavior was shown to depend significantly on the synthesis method. Wet-synthesized powders are significantly less thermally stable compared with those prepared by solid–liquid interaction. Intensive release of carbon oxides from the former was observed at 300 °C, whereas the latter powder was relatively stable up to temperature about 1000 °C.     

Fabrication of Al micro-belts by utilizing electromigration

The fabrication of electromigration induced micro-belts in passivated aluminum films was investigated. The experimental sample was a passivated polycrystalline Al line with a slit and a rectangular hole at the anode end. The micro-belts could be formed at predetermined positions, with their length dependent on the current stress time, and their widths and thicknesses controlled by the size of the hole. It was found that the growth of the micro-belt is affected by the purity of Al, the current density and the current stress time. The experimental results are explained in the discussion.     

Separation of bioconjugated quantum dots using capillary electrophoresis

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was used to separate different bioconjugated CdSe/ZnS quantum dots (QDs). The QD nanocrystals studied were conjugated to the biomolecules streptavidin, biotin, and immunoglobulin G. The bioconjugated QDs showed different electrophoretic mobilities, which appear to depend upon the biomolecule that is attached to the QD and the buffer solution used. The use of a polymeric additive into the CE run buffer improved the resolution of the bioconjugates. Under CE conditions, the interaction between QD bioconjugates containing streptavidin (QDSt) and biotin (QDBi) was monitored. Under a given set of experimental conditions, the fluorescence intensity of QDSt and QDBi emitting light at 655 nm indicated that about 90% of QDBi complexed with 70% of QDSt. A two-color experiment that made use of two different sizes of QD (i.e., 585 and 655 nm) indicated that 30% of the 655 nm QDBi complexed with 53% of the 585 nm QDSt. The use of QDs with different emission properties allows the selective monitoring of two different wavelengths while using one single excitation source. This, in turn, allowed the monitoring of overlapping peaks in the electropherogram when newly formed products resulting from the interaction of the two bioconjugated QDs appeared.