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Analytical and Bioanalytical Chemistry (v.391, #7)

Everything you ever wanted to know about awards by Patricia Ann Mabrouk (pp. 2373-2376).

is Professor of Chemistry and Chemical Biology at Northeastern University (Boston, MA). Her research interests are in chemical education (graduate education, active learning, and undergraduate research), green chemistry, and bioanalytical chemistry.

Daniel S. Sem (Ed.): Spectral techniques in proteomics by Wolf D. Lehmann (pp. 2377-2378).

Peter R. Griffiths, James A. de Haseth: Fourier transform infrared spectrometry (2nd edn.) by Reiner Salzer (pp. 2379-2380).

Thomas Schrader (Ed.): Creative chemical sensor systems by Karin Wöllner (pp. 2381-2382).

Nanostructure in bioanalysis by Takehiko Kitamori (pp. 2383-2383).

Towards single biomolecule handling and characterization by MEMS by Hideyuki F. Arata; Momoko Kumemura; Naoyoshi Sakaki; Hiroyuki Fujita (pp. 2385-2393).

Applications of microelectromechanical systems (MEMS) technology are widespread in both industrial and research fields providing miniaturized smart tools. In this review, we focus on MEMS applications aiming at manipulations and characterization of biomaterials at the single molecule level. Four topics are discussed in detail to show the advantages and impact of MEMS tools for biomolecular manipulations. They include the microthermodevice for rapid temperature alternation in real-time microscopic observation, a microchannel with microelectrodes for isolating and immobilizing a DNA molecule, and microtweezers to manipulate a bundle of DNA molecules directly for analyzing its conductivity. The feasibilities of each device have been shown by conducting specific biological experiments. Therefore, the development of MEMS devices for single molecule analysis holds promise to overcome the disadvantages of the conventional technique for biological experiments and acts as a powerful strategy in molecular biology. Figure Towards single bio molecular handling and characterization by MEMS

Keywords: Microelectromechanical systems; Single molecule; Microheater; Microcontainer; Microtweezers; Microchannel

DNA linearization through confinement in nanofluidic channels by Nicholas Douville; Dongeun Huh; Shuichi Takayama (pp. 2395-2409).

Stretching DNA has emerged as a vital process for studying the physical and biological properties of these molecules. Over the past decade, there has been increasing research interest in utilizing nanoscale fluidic channels to confine and stretch single DNA molecules. Nanofabricated systems for linearizing DNA have revealed new and important insights into the conformation changes of DNA molecules. They also have emerged as innovative techniques for efficiently separating DNA molecules based on size and for physically mapping genetic information along the genome. This review describes physical theories of DNA linearization, current DNA stretching techniques based on nanofabricated channels, and breakthroughs resulting from the use of nanofluidic channels for DNA linearization.

Keywords: DNA linearization; Nanochannels; Confinement; DNA stretching; Nanofluidics; Gene mapping

Photochemical and analytical applications of gold nanoparticles and nanorods utilizing surface plasmon resonance by Ichiro Uechi; Sunao Yamada (pp. 2411-2421).

In recent years, plasmonics has emerged as a promising tool in the fields of analytical chemistry and biochemistry. In particular, surface plasmon resonance at the surfaces of gold nanostructures has led to the development of widespread interest in gold nanoparticles. In this review, we describe some of the recent progress in the manufacture and use of gold nanoparticles, with particular emphasis on gold nanorods. Furthermore, the spectroscopic and photochemical applications of gold nanospheres and nanorods are described.

Keywords: Plasmon; Photochemistry; Surface-enhanced Raman scattering; Evanescent illumination; Molecular thin film; Photoelectric conversion

Highly sensitive restriction enzyme assay and analysis: a review by Liza Lam; Ryota Iino; Kazuhito V. Tabata; Hiroyuki Noji (pp. 2423-2432).

Biological assays at the single molecule level are crucial to fundamental studies of DNA-protein mechanisms. In order to cater for high throughput applications, one area of immense research potential is single-molecule bioassays where miniaturized devices are developed to perform rapid and effective biological reactions and analyses. With the success of various emerging technologies for engineering miniaturized structures down to the nanoscale level, supported by specialized equipment for detection, many investigations in the field of life science that were once thought impossible can now be actively explored. In this review, the significance of downscaling to the single-molecule level is firstly presented in selected examples, with the focus placed on restriction enzyme assays. To determine the effectiveness of single-molecule restriction enzyme reactions, simple and direct analytical methods based on DNA stretching have often been reliably employed. DNA stretching can be realized based on a number of working principles related to the physical forces exerted on the DNA samples. We then discuss two examples of a nanochannel system and a microchamber system where single-molecule restriction enzyme digestion and DNA stretching have been integrated, which possess prospective capabilities of developing into highly sensitive and high-throughput restriction enzyme assays. Finally, we take a brief look at the general trends in technological development in this field by comparing the advantages and disadvantages of performing assays at bulk, microscale and single-molecule levels. Figure Minaturization of Restriction Enzyme Assays and DNA Stretching

Keywords: Bioassays; Single-molecule; Miniaturization; Highly sensitive; Restriction enzyme

Cell electrophoresis on a chip: what can we know from the changes in electrophoretic mobility? by Takanori Akagi; Takanori Ichiki (pp. 2433-2441).

An overview of both experimental and theoretical studies of cell electrophoresis mobility (EPM) over the past fifty years and the relevance of cell EPM measurement are presented and discussed from the viewpoint of exploring the potential use of cell EPM as an index of the biological condition of cells. Physical measurements of the optical and/or electrical properties of cells have been attracting considerable attention as noninvasive cell-evaluation methods that are essential for the future of cell-based application technologies such as cell-based drug screening and cell therapy. Cell EPM, which can be measured in a noninvasive manner by cell electrophoresis, reflects the electrical and mechanical properties of the cell surface. Although the importance of cell EPM has been underestimated for a long time, mostly owing to the technical difficulties associated with its measurement, recent improvements in measurement technology using microcapillary chips have been changing the situation: cell EPM measurement has become more reliable and faster. Recent studies using the automated microcapillary cell electrophoresis system have revealed the close correlation between cell EPM and important biological phenomena including cell cycle, apoptosis, enzymatic treatment, and immune reaction. In particular, the converged EPM distribution observed for synchronized cells has altered the conventional belief that cell EPMs vary considerably. Finding a new significance of cell EPM is likely to lead to noninvasive cell evaluation methods essential for the next-generation of cell engineering.

Keywords: Cell analysis; Microcapillary electrophoresis chip; Electrophoretic mobility; Quality control; Cell cycle; Apoptosis; Immunity

Label-free detection with micro optical fluidic systems (MOFS): a review by A. Q. Liu; H. J. Huang; L. K. Chin; Y. F. Yu; X. C. Li (pp. 2443-2452).

The paper reviews the state-of-art for micro optical fluidic systems (MOFS), or optofluidics, which employs optics and fluidics in a microsystem environment to perform novel functionalities and in-depth analysis in the biophysical area. Various topics, which include the introduction of MOFS in biomedical engineering, the implementation of near-field optics and also the applications of MOFS to biophysical studies, are discussed. Different optical detection techniques, such as evanescent wave, surface plasmon resonance, surface enhanced Raman scattering, resonators and transistors, have been studied extensively and integrated into MOFS. In addition, MOFS also provides a platform for various studies of cell biophysics, such as cell mass determination and cell Young’s modulus measurement. Figure Cell encapsulation and trapping for refractive index measurement in MOFS

Keywords: Micro optical fluidic system; Surface plasmon resonance; Surface enhanced Raman scattering; Resonator; Cell biophysics; Optofluidics

Synergism between particle-based multiplexing and microfluidics technologies may bring diagnostics closer to the patient by S. Derveaux; B. G. Stubbe; K. Braeckmans; C. Roelant; K. Sato; J. Demeester; S. C. De Smedt (pp. 2453-2467).

In the field of medical diagnostics there is a growing need for inexpensive, accurate, and quick high-throughput assays. On the one hand, recent progress in microfluidics technologies is expected to strongly support the development of miniaturized analytical devices, which will speed up (bio)analytical assays. On the other hand, a higher throughput can be obtained by the simultaneous screening of one sample for multiple targets (multiplexing) by means of encoded particle-based assays. Multiplexing at the macro level is now common in research labs and is expected to become part of clinical diagnostics. This review aims to debate on the “added value” we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze, and (c) manipulate the particles are described. Special emphasis is placed on the challenges of integrating currently existing detection platforms for encoded microparticles into microdevices and on promising microtechnologies that could be used to down-scale the detection units in order to obtain compact miniaturized particle-based multiplexing platforms.

Keywords: Bioassays; Biochips/high-throughpout screening; Microfluidics/microfabrication; Encoded particles; Multiplexing; Integrated systems

Semiconductor quantum dots and metal nanoparticles: syntheses, optical properties, and biological applications by Vasudevanpillai Biju; Tamitake Itoh; Abdulaziz Anas; Athiyanathil Sujith; Mitsuru Ishikawa (pp. 2469-2495).

We review the syntheses, optical properties, and biological applications of cadmium selenide (CdSe) and cadmium selenide–zinc sulfide (CdSe–ZnS) quantum dots (QDs) and gold (Au) and silver (Ag) nanoparticles (NPs). Specifically, we selected the syntheses of QDs and Au and Ag NPs in aqueous and organic phases, size- and shape-dependent photoluminescence (PL) of QDs and plasmon of metal NPs, and their bioimaging applications. The PL properties of QDs are discussed with reference to their band gap structure and various electronic transitions, relations of PL and photoactivated PL with surface defects, and blinking of single QDs. Optical properties of Ag and Au NPs are discussed with reference to their size- and shape-dependent surface plasmon bands, electron dynamics and relaxation, and surface-enhanced Raman scattering (SERS). The bioimaging applications are discussed with reference to in vitro and in vivo imaging of live cells, and in vivo imaging of cancers, tumor vasculature, and lymph nodes. Other aspects of the review are in vivo deep tissue imaging, multiphoton excitation, NIR fluorescence and SERS imaging, and toxic effects of NPs and their clearance from the body. Figure Semiconductor quantum dots and metal nanoparticles have extensive applications, e.g., in vitro and in vivo bioimaging

Keywords: Quantum dots; CdSe; CdSe–ZnS; Photoluminescence; Blinking; Nanoparticles; Au; Ag; Plasmon; SERS; In vitro imaging; In vivo imaging

Molecular separation in the lipid bilayer medium: electrophoretic and self-spreading approaches by Hideki Nabika; Baku Takimoto; Kei Murakoshi (pp. 2497-2506).

Molecular separation in a microchannel is a key technology for future miniature devices. Because of growing advances in microfabrication techniques, we now have various choices of microscopic molecular separation systems. Recent progress in this field is reviewed in this manuscript. In particular, the use of the lipid bilayer as a separation medium is highlighted, because of its possible application to the manipulation of relatively small biomaterials such as membrane proteins and lipids. In this context, an approach based on electrophoresis is reviewed for molecular separation in the bilayer. Although the methods based on electrophoresis are effective, we will also focus on their limitation, i.e., only charged molecules can be manipulated. To solve this dilemma, we review new techniques based on the self-spreading nature of the lipid bilayer. In this method, there is no need to input an external field, such as an electric field, to achieve molecular separation. Phenomenological insights into the self-spreading nature and newly proposed molecular separation effects are shown in detail. This novel concept enables us to establish a completely unbiased molecular separation system in future microscopic and nanoscopic devices.

Keywords: Lipid bilayer; Self-spreading; Molecular separation; Nanostructure; Chemical potential

Single-drop analysis of various proteases in a cancer cell lysate using a capillary-assembled microchip by Terence G. Henares; Fumio Mizutani; Ryuichi Sekizawa; Hideaki Hisamoto (pp. 2507-2512).

Single-drop analysis of two different real sample solutions (2 µL) while simultaneously monitoring the activity of two sets of ten different proteases on a single microfluidic device is presented. The device, called a capillary-assembled microchip (CAs-CHIP), is fabricated by embedding square glass sensing capillaries (reagent-release capillaries, RRC) in the polydimethylsiloxane (PDMS) lattice microchannel, and used for that purpose. First, the performance reliability was evaluated by measuring the fluorescence response of twenty caspase-3-sensing capillaries on a single CAs-CHIP, and a relative standard deviation of 1.5–8.2 (% RSD, n = 5 or 10) was obtained. This suggests that precise multiplexed protease-activity sensing is possible by using a single CAs-CHIP with multiple RRCs embedded. Then, using a single CAs-CHIP, real sample analysis of the activity of ten different caspases/proteases in cervical cancer (HeLa) cell lysate treated and untreated with the cell-death-inducer drug, doxorubicin, was simultaneously carried out, and a significant difference in enzyme activity between these two samples was observed. These results suggested the usefulness of the CAs-CHIP in the field of drug discovery. Figure Single drop analysis of two real sample solutions including various different proteases using a single microfluidic device was achieved

Keywords: Capillary-assembled microchip; HeLa cell lysate; Multiplexed protease-activity sensing; Reagent-release capillary

Self-organized ZnO nanorod with photooxidative cell membrane perforation enables large-scale cell manipulation by Takashi K. Saito; Munetoshi Seki; Hitoshi Tabata (pp. 2513-2519).

Various devices have been developed for verification and application of cellular functions in recent years. In our previous study, we found that local oxidation reactions in the cell membrane could produce submicron sizes of reversible membrane perforations in cells, while more than 80% of treated cells were viable even after perforations; therefore, to date, we have attempted some applications of this mechanism and analyzed their feasibility. In the present study, we developed a rod-shaped device in which the function of membrane perforation is added by utilizing a photosensitizer and, using the device, we have attempted to produce membrane perforations in a large number of cells. Zinc oxide nanorods were synthesized on the basis of the vapor–liquid–solid mechanism and α-terthienyl (photosensitizer) was adsorbed onto gold at the top of the rods to add a membrane perforation function. We studied the effect of the oxidation catalytic ability of the rods on rat PC12 cells after pressing and making the rods’ growth side come into contact with the base plate pressed onto the cells in a culture plate followed by photoexcitation of the photosensitizer for a certain period of time. It was revealed that water-soluble fluorescent marker molecules added extracellularly were taken up by the cells when the rods were applied at a pressure of 70 g/cm², with a light intensity of 0.82 W/cm², and with light irradiation for 30 s, as found in the case of the conventional photochemical cell membrane perforation method targeted at a single cell. These results suggest that cell membrane perforation can be successfully achieved in a large number of cells at a time. Figure Large-scale cell membrane perforation process using self-organized nanorods

Keywords: Cell membrane; Microinjection; Photosensitizer; Self-organized material; Zinc oxide

Circular dichroism thermal lens microscope in the UV wavelength region (UV-CD-TLM) for chiral analysis on a microchip by Kazuma Mawatari; Shun Kubota; Takehiko Kitamori (pp. 2521-2526).

We have developed a circular-dichroism thermal lens microscope for UV wavelengths (UV-CD-TLM), for the first time, to realize sensitive chiral analysis on a microchip. Quasi-continuous-wave phase modulation of a pulsed UV laser was used to generate left-circularly polarized light and right-circularly polarized light and to detect the generated TL signal amplitude and phase with a lock-in amplifier. The amplitude and phase were used to determine the concentration and chirality, respectively, of a sample. The basic principle of UV-CD-TLM for chiral analysis on a microchip was verified by measuring aqueous solutions of optically active camphorsulfonic acids (CSA). Lower limits of detection (LOD) were calculated at S/N = 2 and were 8.7 × 10⁻⁴ mol L⁻¹ (ΔA = 5.2 × 10⁻⁶ Abs.) for (+)-CSA and 8.4 × 10⁻⁴ mol L⁻¹ (ΔA = 5.0 × 10⁻⁶ Abs.) for (−)-CSA. In terms of number of molecules, LODs for UV-CD-TLM were calculated to be 8.7 fmol and 8.4 fmol, respectively. This is at least three orders of magnitude lower than previously obtained. The applicability of UV-CD-TLM for chiral analysis on a microchip was verified. Figure Sensitive chiral analysis by thermal lens microscope (TLM)

Keywords: Thermal lens microscope; Circular dichroism effect; Nonfluorescent molecules; Microchip; Chiral analysis

12-Mercaptododecyl β-maltoside-modified gold nanoparticles: specific ligands for concanavalin A having long flexible hydrocarbon chains by Yukari Sato; Teiichi Murakami; Kyoko Yoshioka; Osamu Niwa (pp. 2527-2532).

A simple and highly specific protein detection system using glycoconjugated gold nanoparticles was investigated. This system was based on the aggregation of gold nanoparticles coated with carbohydrate alkanethiols in the presence of corresponding proteins (lectins) that had specific recognition for certain carbohydrates. In order to construct an efficient specific recognition system, maltoside alkanethiol was adopted as an effective sensing modifier having a disaccharide group and a flexible long alkyl chain. The surface modification of gold nanoparticles with maltoside alkanethiol resulted in a shift and broadening (from 520 to 610 nm) of the absorption peak. Monodispersed maltoside-adsorbed gold nanoparticles aggregated with the specific lectin, concanavalin A (Con A). This phenomenon was used to detect the presence of Con A and to estimate concentrations of Con A in sample solutions. The precipitate of the maltoside–gold nanoparticle–Con A mixture was redispersed by addition of methyl α-D-mannopyranoside whose adsorption coefficient is larger than that of maltoside with Con A.

Keywords: Gold nanoparticle; Carbohydrate; Concanavalin A; Self-assembled monolayer; 12-Mercaptododecyl β-maltoside

Highly efficient analysis of underivatized carbohydrates using monolithic-silica-based capillary hydrophilic interaction (HILIC) HPLC by Tohru Ikegami; Kanta Horie; Nabil Saad; Ken Hosoya; Oliver Fiehn; Nobuo Tanaka (pp. 2533-2542).

A polyacrylamide (PAAm)-modified monolithic silica capillary column of increased phase ratio, 200T-PAAm, for hydrophilic interaction liquid chromatography (HILIC) was prepared. The column showed high separation efficiency, with a theoretical plate height H = 7–20 μm at a linear velocity, u = 1–7 mm/s. From a kinetic plot analysis, it was expected that the monolithic column could provide three times faster separation than particle-packed HILIC columns under a pressure limit at 20 MPa. HILIC coupled with electrospray ionization (ESI)–mass spectrometry (HILIC-ESI-MS) using the 200T-PAAm column was employed for the analysis of underivatized carbohydrates to achieve fast and efficient separations of mixtures containing mono-, di-, and trisaccharides within 5 min. Under single MS full scan mode, 200 pg of oligosaccharides was detected by the system. The limit of detection (LOD) of the LC-ESI-MS/MS system was determined using selected reaction monitoring (SRM) to be as low as 3.2 ng/mL (attomol level) for nonreducing saccharides. The system was successfully applied to the detection of disaccharides in extracts of plant, such as corn, soybean, and Arabidopsis thaliana. Figure HILIC-ESI-MS provides a high-efficiency separation and sensitive detection of underivatized carbohydrate oligomers, e.g., the homologs of glucose (1) up to maltoheptaose (7)

Keywords: Monolithic silica; HILIC; Polyacrylamide; Underivatized carbohydrates; HILIC-ESI-MS

A viscosity-tunable polymer for DNA separation by microchip electrophoresis by Daisuke Kuroda; Yong Zhang; Jun Wang; Noritada Kaji; Manabu Tokeshi; Yoshinobu Baba (pp. 2543-2549).

A thermo-responsive separation matrix, consisting of Pluronic F127 tri-block copolymers of poly(ethylene oxide) and poly(propylene oxide), was used to separate DNA fragments by microchip electrophoresis. At low temperature, the polymer matrix was low in viscosity and allowed rapid loading into a microchannel under low pressure. With increasing temperatures above 25°C, the Pluronic F127 solution forms a liquid crystalline phase consisting of spherical micelles with diameters of 17–19 nm. The solution can be used to separate DNA fragments from 100 bp to 1500 bp on poly(methyl methacrylate) (PMMA) chips. This temperature-sensitive and viscosity-tunable polymer provided excellent resolution over a wide range of DNA sizes. Separation is based on a different mechanism compared with conventional matrices such as methylcellulose. To illustrate the separation mechanism of DNA in a Pluronic F127 solution, DNA molecular imaging was performed by fluorescence microscopy with F127 polymer as the separation matrix in microchip electrophoresis. Figure Temperature dependence of the viscosity of 20% w/w Pluronic F127 solution in 1xTBE buffer. Dotted approximates resultant curve.

Keywords: Pluronic F127; Viscosity; Microchip electrophoresis; DNA

Anion exchange silica monolith for capillary liquid chromatography by Jafariah Jaafar; Yuta Watanabe; Tohru Ikegami; Kosuke Miyamoto; Nobuo Tanaka (pp. 2551-2556).

An anion exchange monolithic silica capillary column was prepared by surface modification of a hybrid monolithic silica capillary column prepared from a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS). The surface modification was carried out by on-column copolymerization of N-[3-(dimethylamino)propyl]acrylamide methyl chloride-quaternary salt (DMAPAA-Q) with 3-methacryloxypropyl moieties bonded as an anchor to the silica surface to form a strong anion exchange stationary phase. The columns were examined for their performance in liquid chromatography (LC) and capillary electrochromatography (CEC) separations of common anions. The ions were separated using 50 mM phosphate buffer at pH 6.6. Evaluation by LC produced an average of 30,000 theoretical plates (33 cm column length) for the inorganic anions and nucleotides. Evaluation by CEC, using the same buffer, produced enhanced chromatographic performance of up to ca. 90,000 theoretical plates and a theoretical plate height of ca. 4 μm. Although reduced efficiency was observed for inorganic anions that were retained a long time, the results of this study highlight the potential utility of the DMAPAA-Q stationary phase for anion separations. Figure Micro-LC performance evaluation of a strong anion exchange silica monolith column, 100H-MOP-DMAPAA-Q, 33 cm in length, with a mobile phase of 50 mM phosphate buffer, pH 2.8; linear velocity: u = 1.8 mm/s; UV-Vis detection at 254 nm. Sample solution (5 mg/mL of each component, 4 mL) was injected in split flow injection mode at a split ratio of ca. 1:1900 with a pump flow rate of 1.5 mL/min

Keywords: Anion exchange; Silica monolith; Micro liquid chromatography; Capillary electrochromatography; Inorganic anions

Luminescence-based methods for sensing and detection of explosives by Melissa S. Meaney; Victoria L. McGuffin (pp. 2557-2576).

The detection of explosives and related compounds is important in both forensic and environmental applications. Luminescence-based methods have been widely used for detecting explosives and their degradation products in complex matrices. Direct detection methods utilize the inherent fluorescence of explosive molecules or the luminescence generated from chemical reactions. Direct detection methods include high-energy excitation techniques such as gamma-ray and x-ray fluorescence, detection of decomposition products by fluorescence or chemiluminescence, and detection following reduction to amines or another reaction to produce fluorescent products from the explosive. Indirect detection methods utilize the interference caused by the presence of explosive compounds with traditional processes of fluorescence and fluorescence quenching. Indirect detection methods include quenching of solution-phase, immobilized, and solid-state fluorophores, displacement of fluorophores, fluorescence immunoassay, and reactions that produce fluorescent products other than the explosive. A comprehensive review of these methods is presented.

Keywords: Luminescence; Fluorescence; Fluorescence quenching; Chemiluminescence; Explosives

Rapid, single-step nucleic acid detection by Kyle A. Cissell; Sean Campbell; Sapna K. Deo (pp. 2577-2581).

A rapid detection method for nucleic acid based on bioluminescence resonance energy transfer (BRET) from the luminescence donor Renilla luciferase to an acceptor quantum dot upon oligonucleotide probe hybridization has been developed. Utilizing a competitive assay, we detected the target nucleic acid by correlating the BRET signal with the amount of target present in the sample. This method allows for the detection of as little as 4 pmol (20 nM) of nucleic acid in a single-step, homogeneous format both in vitro in a buffer matrix as well as in a cellular matrix. Using this method, one may perform nucleic acid detection in as little as 30 min, showing much improvement over time-consuming blotting methods and solid-phase methods which require multiple wash steps to remove unbound probe. This is the first report on the use of quantum dots as a BRET acceptor in the development of a nucleic acid hybridization assay.

Keywords: Bioluminescence resonance energy transfer; Renilla luciferase; Quantum dots; Nucleic acid detection; Nucleic acid hybridization

Rapid, single-step nucleic acid detection by Kyle A. Cissell; Sean Campbell; Sapna K. Deo (pp. 2577-2581).

Keywords: Bioluminescence resonance energy transfer; Renilla luciferase; Quantum dots; Nucleic acid detection; Nucleic acid hybridization

High-resolution ICP–MS determination of Ti, V, Cr, Co, Ni, and Mo in human blood and urine of patients implanted with a hip or knee prosthesis by Alejandro Sarmiento-González; Juan Manuel Marchante-Gayón; José María Tejerina-Lobo; José Paz-Jiménez; Alfredo Sanz-Medel (pp. 2583-2589).

The main components (Ti, V, Cr, Co, Ni, and Mo) of metallic alloys currently used in hip and knee articular prostheses have been simultaneously determined in human whole blood and urine of implanted people by a (HR)-ICP–MS method previously developed in our laboratory. The determination of those elements has been carried out in patients with knee and hip prosthesis and in a group of pre-operation patients without any metallic device in their bodies, used as controls, demonstrating the usefulness of this technique to perform multielement analysis at ppt levels in complex matrices. The concentrations of V, Cr, Co, Ni, and Mo in urine and blood of implanted people turned out to be very similar to those obtained in control patients. However, raised Ti levels could be found both in urine and blood of patients with articular prostheses made or coated with a titanium alloy (Ti₆Al₄V).

Keywords: HR-ICP–MS; Metals release; Hip and knee articular prosthesis; Blood and urine samples

Estimating relative carbonyl levels in muscle microstructures by fluorescence imaging by Juan Feng; Marian Navratil; LaDora V. Thompson; Edgar A. Arriaga (pp. 2591-2598).

The increase in the levels of protein carbonyls, biomarkers of oxidative stress, appears to play an important role in aging skeletal muscle. However, the exact distributions of carbonyls among various skeletal muscle microstructures still remain largely unknown, partly owing to the lack of adequate techniques to carry out these measurements. This report describes an immunohistochemical approach to determine the relative abundance of carbonyls in the intermyofibrillar mitochondria (IFM), the subsarcolemmal mitochondria (SSM), the cytoplasm, and the extracellular space of skeletal muscle. These morphological features were defined by labeling the nucleus, the Z-lines, and mitochondria. Carbonyls were detected by derivatization with dinitrophenylhydrazine followed by labeling with an Alexa 488-labeled anti-dinitrophenyl primary antibody. Alexa 488 fluorescence (green) in different fiber microstructures was used to estimate the relative abundance of carbonyls. On the basis of the samples examined, preliminary results suggest that the most dramatic age-related changes in carbonyl levels occur in the extracellular space, followed in a decreasing order by SSM, IFM, and the cytoplasm. These observations were confirmed in the soleus and semimembranosus muscles composed predominantly of type I and type II fibers, respectively. This approach could easily be extended to the investigation of carbonyl levels in other muscles (composed of mixed skeletal muscle fiber types) or other tissues in which protein carbonyls are present. Figure Imaging of Labeled Carbonyls in Rat Skeletal Muscle

Keywords: Protein carbonyls; Aging; Skeletal muscle microstructures; Fluorescence microscopy; Mitochondria

Analysis of large oxygenated and nitrated polycyclic aromatic hydrocarbons formed under simulated diesel engine exhaust conditions (by compound fingerprints with SPE/LC-API-MS) by Christoph Adelhelm; Reinhard Niessner; Ulrich Pöschl; Thomas Letzel (pp. 2599-2608).

The analysis of organic compounds in combustion exhaust particles and the chemical transformation of soot by nitrogen oxides are key aspects of assessment and mitigation of the climate and health effects of aerosol emissions from fossil fuel combustion and biomass burning. In this study we present experimental and analytical techniques for efficient investigation of oxygenated and nitrated derivatives of large polycyclic aromatic hydrocarbons (PAHs), which can be regarded as well-defined soot model substances. For coronene and hexabenzocoronene exposed to nitrogen dioxide under simulated diesel exhaust conditions, several reaction products with high molecular mass could be characterized by liquid chromatography-atmospheric pressure chemical (and photo) ionization-mass spectrometry (LC-APCI-MS and LC-APPI-MS). The main products of coronene contained odd numbers of nitrogen atoms (m/z 282, 256, 338), whereas one of the main products of hexabenzocoronene exhibited an even number of nitrogen atoms (m/z 391). Various reaction products containing carbonyl and nitro groups could be tentatively identified by combining chromatographic and mass spectrometric information, and changes of their relative abundance were observed to depend on the reaction conditions. This analytical strategy should highlight a relatively young technique for the characterization of various soot-contained, semi-volatile, and semi-polar reaction products of large PAHs. Figure LC-APCI-MS analysis of nitrated coronene (and HBC): Total-Ion-Chromatogram (TIC), Extracted Ion Chromatograms (EICs) and corresponding mass spectrum (top).

Keywords: LC-API-MS; High-performance liquid chromatography; Mass spectrometry; Oxidation; Nitration; PAH

Micro-plasma: a novel ionisation source for ion mobility spectrometry by Wolfgang Vautz; Antje Michels; Joachim Franzke (pp. 2609-2615).

Ion mobility spectrometry is an analytical method for identification and quantification of gas-phase analytes in the ppb_v-ppt_v range. Traditional ionisation methods suffer from low sensitivity (UV light), lack of long-term stability (partial discharge), or legal restrictions when radioactive sources are used. A miniaturised helium plasma was applied as ionisation source in an ion mobility spectrometer (IMS). Experiments were carried out to compare plasma IMS with β-radiation IMS. It could be demonstrated that the plasma IMS is characterised by higher sensitivity and selectivity than β-radiation ionisation. Plasma IMS is approximately 100 times more sensitive than the β-radiation IMS. Furthermore, variable sensitivity can be achieved by variation of the helium flow and the electric field of the plasma, and variable selectivity can be achieved by changing the electric field of the IMS. The experimental arrangement, optimisation of relevant conditions, and a typical application are presented in detail. Figure Micro-plasma used in ion mobility spectrometry

Keywords: Ion mobility spectrometry; Ionisation sources; Plasma; Sensitivity; Selectivity; Helium; VOC

Photofragmentation of nitro-based explosives with chemiluminescence detection by Maria Pamela P. Monterola; Benjamin W. Smith; Nicolò Omenetto; James D. Winefordner (pp. 2617-2626).

A simple, fast, reliable, sensitive and potentially portable explosive detection device was developed employing laser photofragmentation (PF) followed by heterogeneous chemiluminescence (CL) detection. The PF process involves the release of NO_{x(x = 1,2)} moieties from explosive compounds such as TNT, RDX, and PETN through a stepwise excitation–dissociation process using a 193 nm ArF laser. The NO_{x(x = 1,2)} produced upon PF is subsequently detected by its CL reaction with basic luminol solution. The intensity of the CL signal was detected by a thermoelectrically cooled photomultiplier tube with high quantum efficiency and negligible dark current counts. The system was able to detect trace amounts of explosives in various forms in real time under ambient conditions. Detection limits of 3 ppbv for PETN, 2 ppbv for RDX, and 34 ppbv for TNT were obtained. It was also demonstrated that the presence of PETN residue within the range of 61 to 186 ng/cm² can be detected at a given signal-to-background ratio of 10 using a few microjoules of laser energy. The technique also demonstrated its potential for the direct analysis of trace explosive in soil. An LOD range of 0.5–4.3 ppm for PETN was established, which is comparable to currently available techniques. Figure Photofragmentation–chemiluminescence detector

Keywords: Nitro-based explosives; Photofragmentation; Chemiluminescence; TNT; RDX; PETN

Synthesis, characterization, and high-performance liquid chromatographic evaluation of C₁₄ stationary phases containing branched and unbranched alkyl groups by Benjamin Dietrich; Karsten Holtin; Marc Bayer; Volker Friebolin; Max Kühnle; Klaus Albert (pp. 2627-2633).

Reversed-phase materials with branched and unbranched alkyl groups were prepared by modifying porous, spherical silica gel in a two-step reaction—immobilization of a trifunctional alkoxysilane (3-glycidoxypropyltrimethoxysilane) on the silica surface followed by reaction with a branched and an unbranched octanoic acid. The chromatographic sorbents were characterized by solid-state ²⁹Si and ¹³C NMR spectroscopy. The chromatographic behaviour of the stationary phases was evaluated by use of a test mixture according to the Standard Reference Material 870 set from the US National Institute of Standards and Technology, in order to study the effect of branched and unbranched alkyl chains.

Keywords: C₁₄ stationary phases; Solid-state CP/MAS NMR spectroscopy; HPLC; Branching effect of alkyl chains

Development of a methodology utilizing gas chromatography ion-trap tandem mass spectrometry for the determination of low levels of caffeine in surface marine and freshwater samples by Sergei S. Verenitch; Asit Mazumder (pp. 2635-2646).

A methodology for monitoring low level of caffeine in aqueous samples via gas chromatography coupled with an ion-trap tandem mass spectrometry detection system (IT-MS/MS) was developed. Four IT-MS/MS operating parameters, including the collision-induced dissociation (CID) voltage, the excitation time (ET), the isolation time (IT) and the maximum ionization time (MIT) were optimized in order to maximize the sensitivity of the IT-MS/MS technique towards the analyte and its isotope-labeled standard. After optimization, a limit of detection of 500 fg μl⁻¹ with S/N = 3 was achieved. Taking into account blank values and the matrix background, a method detection limit of 1.0–2.0 ng l⁻¹ was derived and applied to all of the samples analyzed in the study. Various mass spectrometric conditions have been applied to caffeine and its trimethyl-¹³C-labeled standard to elucidate fragmentation pathways for new and commonly occurring product ions observed in the collision-induced dissociation (CID) spectra produced by the ion trap. Ion structures and fragmentation pathway mechanisms have been proposed and compared with previously published data. An isotope dilution method using ¹³C-labeled caffeine as a surrogate internal standard was employed to determine and correct the recovery of native caffeine in water samples. The developed methodology has been applied for the determination of caffeine in surface marine and freshwater samples collected on the west coast of Vancouver Island in British Columbia, Canada. The results obtained for the marine water samples indicated a wide variation in the level of caffeine, ranging from 4.5 to 149 ng l⁻¹, depending on the location of the sampling site within the inlet. The concentrations of caffeine in samples from lakes associated with various residential densities ranged from ND to 6.5, 1.8 to 10.4 and 6.1 to 21.7 ng l⁻¹ for low, moderate and high residential densities, respectively.

Keywords: Caffeine; Surface water; Ion trap GC-MSMS

Determination (monitoring) of PAHs in surface waters: why an operationally defined procedure is needed by Lidia Wolska (pp. 2647-2652).

The results of interlaboratory studies on the determination of selected PAHs in samples of pure water and water containing suspended matter are presented and discussed in this study. Determinations were performed by independent analytical laboratories which used different sample-preparation procedures, i.e. liquid-liquid extraction and solid-phase extraction with columns and speedisks. The study indicates that the results of PAHs determination depend on the type of isolation technique and the final determination procedure used by a given laboratory. Differences among the determined concentrations of specific PAHs reached 700%. In this work it has been shown that modern analysis does not offer isolation techniques for PAHs which would secure their speciation in the aquatic environment.

Keywords: PAH; Surface water; Analytical procedures; Suspended particulate matter; Interlaboratory studies

A highly sensitive and specific polyclonal antibody-based enzyme-linked immunosorbent assay for detection of antibiotic olaquindox in animal feed samples by Dan Zhao; Li He; Chun Pu; Anping Deng (pp. 2653-2661).

The use of olaquindox (OLA) as an additive in animal feedstuffs has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for determination of OLA in animal feed samples was developed. OLA was activated by N′N-carbonyldiimidazole and coupled with bovine serum albumin (BSA) and ovalbumin (OVA). It was found that the sensitivity and specificity of the two antisera were very similar, with the IC₅₀ values of 16 ng mL⁻¹ and 19 ng mL⁻¹, respectively. Cross-reactivity was less than 35% for four structurally related compounds and no recognition of five other antibiotics was observed. The better antiserum I was selected for further experiments, for example testing stability, solvent effect, accuracy, and precision. The IC₅₀ value for eight standard curves was in the range 12–18 ng mL⁻¹ and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.31 ± 0.11 ng mL⁻¹. The ELISA tolerated 5% methanol without significant influence on IC₅₀ value. The recoveries of spiked OLA in five different animal feed types including auxin, pig complex feed, fish complex feed, broiler concentrated feed, and pig premix feed were in the range 88.3–119.0% and the intra-assay relative standard deviation (RSD) was within 4.7–33.5% (n = 3). The ELISA for unspiked feed samples was confirmed by high-performance liquid chromatography (HPLC), with a high correlation coefficient of 0.9862 (n = 5). The proposed ELISA could be a feasible quantitative/screening method for OLA analysis in feed samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput, and low expense. Figure Polyclonal antibody based ELISA for detection of olaquindox

Keywords: Olaquindox; Enzyme-linked immunosorbent assay; Polyclonal antibody; Animal feed samples

Analysis of amino acids without derivatization in barley extracts by LC-MS-MS by Björn Thiele; Kerstin Füllner; Nadine Stein; Marco Oldiges; Arnd J. Kuhn; Diana Hofmann (pp. 2663-2672).

A method has been developed for quantification of 20 amino acids as well as 13 ¹⁵N-labeled amino acids in barley plants. The amino acids were extracted from plant tissues using aqueous HCl–ethanol and directly analyzed without further purification. Analysis of the underivatized amino acids was performed by liquid chromatography (LC)–electrospray ionization (ESI) tandem mass spectrometry (MS-MS) in the positive ESI mode. Separation was achieved on a strong cation exchange column (Luna 5µ SCX 100Å) with 30 mM ammonium acetate in water (solvent A) and 5% acetic acid in water (solvent B). Quantification was accomplished using d ₂-Phe as an internal standard. Calibration curves were linear over the range 0.5–50 µM, and limits of detection were estimated to be 0.1–3.0 µM. The mass-spectrometric technique was employed to study the regulation of amino acid levels in barley plants grown at 15 °C uniform root temperature (RT) and 20–10 °C vertical RT gradient (RTG). The LC-MS-MS results demonstrated enhanced concentration of free amino acids in shoots at 20–10 °C RTG, while total free amino acid concentration in roots was similarly low for both RT treatments. ¹⁵NO₃ ⁻ labeling experiments showed lower ¹⁵N/¹⁴N ratios for Glu, Ser, Ala and Val in plants grown at 20–10 °C RTG compared with those grown at 15 °C RT.

Keywords: Liquid chromatography–mass spectrometry; Amino acids; Strong cation exchange column; Plant metabolism

Octadecylsilane-modified silicas in the adsorption of toluene by Rodrigo Brambilla; Carolina F. Pinto; Márcia S. L. Miranda; João H. Z. dos Santos (pp. 2673-2681).

A series of octadecylsilane-modified silicas were prepared by sol-gel and grafting methods. Carbon contents and octadecyl chain conformations were shown to depend on the preparative route. Grafting engenders a low carbon content and a liquid-like chain conformation, while the sol-gel method affords a much higher carbon content and a crystalline conformation. The relationships between the toluene adsorption of the hybrid silicas and their chain conformations, their carbon contents and their textural characteristics are discussed. These sorbents, when used in combination with ultraviolet diffuse reflectance spectroscopy (UV DRS), can be employed as a rapid screening method for detection of aromatic compounds in water and air environmental matrices. Figure Octadecylsilane-modified silicas in the adsorption of toluene

Keywords: Hybrid silicas; Octadecylsilsane; Adsorption; Diffuse reflectance spectroscopy

A liquid chromatography method using a monolithic column for the determination of corticoids in animal feed and animal feeding water by R. Muñiz-Valencia; S. G. Ceballos-Magaña; R. Gonzalo-Lumbreras; A. Santos-Montes; R. Izquierdo-Hornillos (pp. 2683-2691).

An HPLC-DAD method for determining corticoids in calf feed and in animal feeding water samples using a monolithic column has been developed and validated. The method optimization included the study of binary mobile phases of water and acetonitrile. The optimum separation was achieved at 40 °C, with acetonitrile:H₂O 29:71 v/v used as mobile phase and a 3 ml/min flow-rate, which resulted in their separation in about 5 min. Two reported sample procedures were applied to feed and for animal feeding water samples prior to HPLC. Method validation was carried out according to the EU criteria established for quantitative screening methods. The results indicate that this method is highly specific, reproducible and accurate. The proposed method was found to be robust and unaffected by small variations in the extraction procedure and in HPLC conditions. The developed method for the determination of corticoids in feed and water samples was also found to be suitable for different kinds of feeds and waters.

Keywords: Corticoids; Growth-promoting agents; Monolithic column; Animal feed; Animal feeding water

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Analytical and Bioanalytical Chemistry (v.391, #7)