Check out our New Publishers' Select for Free Articles

Analytical and Bioanalytical Chemistry (v.402, #5)

Surface architectures for analytical purposes by Luigia Sabbatini; Luisa Torsi (pp. 1737-1738).

has been full professor of Analytical Chemistry at the University of Bari Aldo Moro since 1986. Currently Advisory Board member of Analytical and Bioanalytical Chemistry, in the past she served for many years as a member of the Editorial Board of the Journal of Electron Spectroscopy and Related Phenomena. She has been Head of the Chemistry Department of the University of Bari Aldo Moro (2005–2010), President of the Laboratory for Diagnostics in Cultural Heritage (2005–2010), President of the Analytical Division of Italian Chemical Society (1997–2000), and coordinator of several national and regional projects. She is the author of approximately one hundred and fifty ISI papers and co-inventor of several patents.Her main scientific interests concern the development and surface characterization of application-oriented innovative materials. Her field of expertise is surface spectroscopy, in particular X-ray photoelectron spectroscopy (XPS), the analytical capabilities of which have been fully exploited in determination of the chemical composition of materials properly tailored for biomedical, microelectronics, and sensors application and in investigations of works of art. ( www.luisatorsi.info ) has been full professor of Analytical Chemistry at the University of Bari Aldo Moro since 2005. She is the 2010 E.H. Merck prize winner for Analytical Science (the first woman to be awarded the prize). Her main research interests are functional materials/nanostructures and electronic devices for chemical and biological sensing. Author of 86 ISI papers, including Science and Nature Materials, she is also co-inventor of several patents. Her work has gathered more than 3500 citations resulting in an h-index of 29. Awarded research funding comprises European contracts, and national and regional projects. Professor Torsi is coordinating an ITN Marie Curie European network named FlexSMELL—Gas Sensors on Flexible Substrates for Wireless Applications ( www.flexsmell.eu ) and she is also principal investigator in the STREP proposal BioEGOFET—Electrolyte-Gated Organic Field-Effect BIOsensors ( www.bioegofet.eu ). She is currently member of the Central Council of the Italian Chemical Society and of the Executive Committee of the European Material Research Society (E-MRS).

Surface-attached sensors for cation and anion recognition by Nicholas H. Evans; Habibur Rahman; Jason J. Davis; Paul D. Beer (pp. 1739-1748).

The development of surface-attached sensors for cationic and anionic guests is of intense current research interest. In addition to the environmental flexibility, robustness and reusability of such devices, surface-confined sensors typically exhibit an amplified response to target analytes owing to preorganization of the receptor. Whereas redox-active cations may be sensed by studying the cyclic voltammetry of host–guest systems containing ion-selective receptors attached to an appropriate electrode, redox-inactive ionic species require the use of electrochemical impedance spectroscopy, with appropriately functionalized electrodes and redox probes. Alternatively, receptors may be constructed that incorporate an electrochemical or optical reporter group within their structure to provide a macroscopic response to the presence of an ionic guest. This critical review seeks to present an up-to-date, although necessarily selective, account of the progress in the field, and provides insights into possible future developments, including the utilization of receptor–nanoparticle conjugates and mechanically interlocked receptors.

Keywords: Anions; Cations; Electrochemistry; Fluorescence; Ion recognition; Self-assembled monolayers

Layer-by-layer construction of protein architectures through avidin–biotin and lectin–sugar interactions for biosensor applications by Shigehiro Takahashi; Katsuhiko Sato; Jun-ichi Anzai (pp. 1749-1758).

In this review, the preparation and properties of protein architectures constructed by layer-by-layer (LbL) deposition through avidin–biotin and concanavalin A (Con A)–sugar interactions are discussed in relation to their use for optical and electrochemical biosensors. LbL films can be constructed through the alternate deposition of avidin and biotin-labeled enzymes on the surfaces of optical probes and electrodes. The enzymes retain their catalytic activity, resulting in the formation of optical and electrochemical biosensors. Alternatively, Con A can be used to construct enzyme-containing LbL films and microcapsules using sugar-labeled enzymes. Some enzymes such as glucose oxidase and horseradish peroxidase can be used for this purpose without labeling with sugar, because these enzymes contain intrinsic hydrocarbon chains on their molecular surfaces. The Con A/enzyme LbL architectures were successfully used to develop biosensors sensitive to specific substrates of the enzyme. In addition, Con A-based films can be used for the optical and electrochemical detection of sugars. Figure Sugar-induced decomposition of Con A/glycogen LbL film for the electrochemical sugar sensing

Keywords: Layer-by-layer film; Protein architecture; Microcapsule; Biosensor; Avidin–biotin; Lectin–sugar

Functionalized gold nanoparticles for ultrasensitive DNA detection by Laura Maria Zanoli; Roberta D’Agata; Giuseppe Spoto (pp. 1759-1771).

A major challenge in the area of DNA detection is the development of rapid methods that do not require polymerase chain reaction (PCR) amplification of the genetic sample. The PCR amplification step increases the cost of the assay, the complexity of the detection, and the quantity of DNA required for the assay. In this context, methods that are able to perform DNA analyses with ultrasensitivity have recently been investigated with the aim of developing new PCR-free detection protocols. Functionalized gold nanoparticles have played a central role in the development of such methods. Here, possibilities offered by functionalized gold nanoparticle in the ultrasensitive detection of DNA are discussed. The different functionalization protocols available for gold nanoparticles and the principal DNA detection methods that are able to detect DNA at the femtomolar to attomolar level are presented.

Keywords: DNA detection; Gold nanoparticles; PCR-free; Optical detection; Electrochemical detection; SPR; QCM; Scanometric detection

Electrochemical plasmonic sensors by Andreas B. Dahlin; Bernd Dielacher; Prayanka Rajendran; Kaori Sugihara; Takumi Sannomiya; Marcy Zenobi-Wong; Janos Vörös (pp. 1773-1784).

The enormous progress of nanotechnology during the last decade has made it possible to fabricate a great variety of nanostructures. On the nanoscale, metals exhibit special electrical and optical properties, which can be utilized for novel applications. In particular, plasmonic sensors including both the established technique of surface plasmon resonance and more recent nanoplasmonic sensors, have recently attracted much attention. However, some of the simplest and most successful sensors, such as the glucose biosensor, are based on electrical readout. In this review we describe the implementation of electrochemistry with plasmonic nanostructures for combined electrical and optical signal transduction. We highlight results from different types of metallic nanostructures such as nanoparticles, nanowires, nanoholes or simply films of nanoscale thickness. We briefly give an overview of their optical properties and discuss implementation of electrochemical methods. In particular, we review studies on how electrochemical potentials influence the plasmon resonances in different nanostructures, as this type of fundamental understanding is necessary for successful combination of the methods. Although several combined platforms exist, many are not yet in use as sensors partly because of the complicated effects from electrochemical potentials on plasmon resonances. Yet, there are clearly promising aspects of these sensor combinations and we conclude this review by discussing the advantages of synchronized electrical and optical readout, illustrating the versatility of these technologies.

Keywords: Plasmon; Electrochemistry; Nanostructure; Sensor; Electrical; Optical

Staying alive: new perspectives on cell immobilization for biosensing purposes by Elisa Michelini; Aldo Roda (pp. 1785-1797).

Intact living cells, because of their simplicity of use and their ability to provide highly valuable functional information, are well suited to biosensing applications. Cells can be genetically engineered by introduction of reporter proteins, modified to achieve analyte selectivity for their sensing capabilities, and connected to a transducer to obtain whole-cell biosensors. These bioanalytical features are increasingly attracting attention in the pharmaceutical, environmental, medical, and industrial fields. Whole-cell biosensors based on different recognition elements and transduction mechanisms have been also incorporated into portable devices and, with recent advances in micro and nanofabrication and microfluidics technology, miniaturized to achieve single-cell level analysis. Cell immobilization, widely used in, for example, microbial biofermentors or bioremediation systems, is now emerging as an appealing way of integrating whole-cell biosensors into devices, to maintain long-term cell viability, to increase the reproducibility of the cell’s response, and to avoid the spread of genetically modified cells into the environment, the latter being very important when devices are used for analysis in the field. A plethora of materials and functionalized surfaces have been proposed for immobilization of microbial or mammalian cells, each one having peculiar advantages and limitations. This critical review highlights and discusses recent trends, together with selected bioanalytical applications of immobilized viable cells. In particular the review focuses on some aspects that seem to hold great promise for future applications of immobilized cells, spanning from microbial biosensors to microbial biofilms, cell microarrays, and single-cell analysis. Figure Most promising applications of immobilized cells: portable devices with microbial biosensors, microbial biofilms, microfluidic devices and surfaces functionalized for mammalian cell immobilization

Keywords: Bioanalytical methods; Biosensors; Cell systems/single-cell analysis; Biomaterials; Biochips/high-throughput screening; Bioassays

Microcantilevers and organic transistors: two promising classes of label-free biosensing devices which can be integrated in electronic circuits by Serafina Cotrone; Damiana Cafagna; Stefania Cometa; Elvira De Giglio; Maria Magliulo; Luisa Torsi; Luigia Sabbatini (pp. 1799-1811).

Most of the success of electronic devices fabricated to actively interact with a biological environment relies on the proper choice of materials and efficient engineering of surfaces and interfaces. Organic materials have proved to be among the best candidates for this aim owing to many properties, such as the synthesis tunability, processing, softness and self-assembling ability, which allow them to form surfaces that are compatible with biological tissues. This review reports some research results obtained in the development of devices which exploit organic materials’ properties in order to detect biologically significant molecules as well as to trigger/capture signals from the biological environment. Among the many investigated sensing devices, organic field-effect transistors (OFETs), organic electrochemical transistors (OECTs) and microcantilevers (MCLs) have been chosen. The main factors motivating this choice are their label-free detection approach, which is particularly important when addressing complex biological processes, as well as the possibility to integrate them in an electronic circuit. Particular attention is paid to the design and realization of biocompatible surfaces which can be employed in the recognition of pertinent molecules as well as to the research of new materials, both natural and inspired by nature, as a first approach to environmentally friendly electronics. Figure Representative scheme of biorecognition in Organic Transistor and Microcantilever devices

Keywords: Organic field-effect transistors (OFETs); Organic electrochemical transistors (OECTs); Microcantilevers; Sensors; Biocompatibility

Advances in organic transistor-based biosensors: from organic electrochemical transistors to electrolyte-gated organic field-effect transistors by Loïg Kergoat; Benoît Piro; Magnus Berggren; Gilles Horowitz; Minh-Chau Pham (pp. 1813-1826).

Organic electronics have, over the past two decades, developed into an exciting area of research and technology to replace classic inorganic semiconductors. Organic photovoltaics, light-emitting diodes, and thin-film transistors are already well developed and are currently being commercialized for a variety of applications. More recently, organic transistors have found new applications in the field of biosensors. The progress made in this direction is the topic of this review. Various configurations are presented, with their detection principle, and illustrated by examples from the literature. Figure Electrolyte-Gated OFET (EGOFET) architecture. EGOFETs differ from OFETs, as in OECTs, in that the gate is separated from the semiconductor by an electrolyte. This allows low voltage operation compared with OFETs gated via solid dielectrics. The red circle indicates the interface involved in the detection of biomolecules, when water is used as electrolyte.

Keywords: Organic thin-film transistors; Electrolyte-gated transistors; Water-gated transistors; Biosensors

MIP sensors – the electrochemical approach by Cosimino Malitesta; Elisabetta Mazzotta; Rosaria A. Picca; Alessandro Poma; Iva Chianella; Sergey A. Piletsky (pp. 1827-1846).

This review highlights the importance of coupling molecular imprinting technology with methodology based on electrochemical techniques for the development of advanced sensing devices. In recent years, growing interest in molecularly imprinted polymers (MIPs) in the preparation of recognition elements has led researchers to design novel formats for improvement of MIP sensors. Among possible approaches proposed in the literature on this topic, we will focus on the electrosynthesis of MIPs and on less common hybrid technology (e.g. based on electrochemistry and classical MIPs, or nanotechnology). Starting from the early work reported in this field, an overview of the most innovative and successful examples will be reviewed.

Keywords: Molecularly imprinted polymers; Electrochemistry; Sensors; Nanotechnology

Electrochemical release of hepatocyte-on-hydrogel microstructures from ITO substrates by Sunny S. Shah; Mihye Kim; Elena Foster; Tam Vu; Dipali Patel; Li-Jung Chen; Stanislav V. Verkhoturov; Emile Schweikert; Giyoong Tae; Alexander Revzin (pp. 1847-1856).

This paper describes a novel platform that utilizes micropatterning and electrochemistry to release cells-on-hydrogel microstructures from conductive indium tin oxide (ITO) substrates. In this approach, UV photopolymerization was employed to micropattern heparin-based hydrogels onto glass substrates containing ITO electrodes. ITO/glass substrates were first functionalized with acrylated silane to promote attachment of hydrogel structures. The surfaces containing hydrogel micropatterns were further functionalized with poly(ethylene glycol) thiol, rendering the regions around the hydrogel structures non-fouling to proteins and cells. After incubating surfaces with collagen (I), primary rat hepatocytes were shown to selectively attach on top of the hydrogel and not on surrounding glass/ITO regions. Electrical activation of specific ITO electrodes (−1.8 V vs. Ag/AgCl reference) was then used to release cells-on-hydrogel microstructures from the substrate. Immunostaining and reverse transcription polymerase chain reaction analysis of albumin, an important indicator of hepatic function, showed that the hepatocyte-on-hydrogel microstructures released from the surface maintained their function at levels similar to hepatocytes remaining on the culture substrate. In the future, switchable conductive substrates described here may be to collect cell samples at different time points and may also be used for harvesting cell-carrying vehicles for transplantation studies. Figure On-cue spatially controlled release of hepatocytes residing on heparin gel microstructures

Keywords: Hepatocytes; Micropatterned surfaces; Cell sorting; Electrochemical release; Heparin-based hydrogel

Stepan Podzimek: Light scattering, size exclusion chromatography and asymmetric flow field flow fractionation. Powerful tools for the characterization of polymers, proteins and nanoparticles by André M. Striegel (pp. 1857-1858).

TOF-SIMS imaging of halide/thiocyanate anions and hydrogen sulfide in mouse kidney sections using silver-deposited plates by Noriyuki Akahoshi; Itsuko Ishizaki; Masayuki Naya; Toshihiko Maekawa; Shougo Yamazoe; Tadashi Horiuchi; Mayumi Kajimura; Yoshiharu Ohashi; Makoto Suematsu; Isao Ishii (pp. 1859-1864).

In vivo imaging of reactive small molecule metabolites with high spatial resolution and specificity could give clues to understanding pathophysiology of various diseases. We herein applied time of flight-secondary ion mass spectrometry (TOF-SIMS) to newly developed silver-deposited plates that were stamped on mouse tissues, and succeeded in visualization of halide (Cl⁻, Br⁻, and I⁻) and pseudohalide thiocyanate (SCN⁻) anions, a class of substrates for neutrophils/eosinophil peroxidases to produce hypohalous acids (HOX/OX⁻ mixture; X: (pseudo)halides), as well as hydrogen sulfide (H₂S). Forty-micrometer frozen mouse kidney sections on cover glasses were attached to 37 °C preheated silver-deposited plates and incubated at −10 °C for 1 h. After sputter cleaning to remove surface contaminants, the plates were analyzed by TOF-SIMS to identify distribution of Br⁻, AgBr ₂ ⁻ , I⁻, AgI ₂ ⁻ , SCN⁻, as well as S²⁻ and AgS⁻ as products of tissue-derived H₂S. Br⁻, AgBr ₂ ⁻ , I⁻, and SCN⁻ anions were mainly distributed in core regions including the inner medulla and inner stripe of the outer medulla (except for I⁻), rather than outer regions such as the cortex and outer stripe of the outer medulla. AgI ₂ ⁻ anion was spread over the whole kidney, although its levels were relatively low. In contrast, S²⁻ and AgS⁻ anions were mainly present in the outer regions. To our knowledge, this is the first imaging study to reveal the distribution of (pseudo)halides and H₂S in animal tissue sections.

Keywords: Halide; Thiocyanate; Hydrogen sulfide; Sliver-deposited plate; TOF-SIMS; Imaging

Determination of ethyl glucuronide in nails by liquid chromatography tandem mass spectrometry as a potential new biomarker for chronic alcohol abuse and binge drinking behavior by Luca Morini; Mario Colucci; Maria Giovanna Ruberto; Angelo Groppi (pp. 1865-1870).

A liquid chromatography tandem mass spectrometry method for ethyl glucuronide (EtG) detection and quantification in nails was developed and fully validated. Nails were extracted in 700 μL double-distilled water. EtG-d ₅ was used as an internal standard. Reversed-phase separation was obtained with an isocratic mobile phase composed of 0.1% formic acid and acetonitrile (99:1) for 10 min. Quantification was performed by multiple reaction monitoring of two transitions per compound (EtG and internal standard). The assay was linear from 10 to 500 pg/mg. Validation parameters were studied at three different quality control levels (10, 50, and 300 pg/mg). Intraday, interday, and total imprecision had a coefficient of variation of less than 9.5%. Ion suppression and ion enhancement were negligible (less than 20%). No carryover was detected. The method was applied to several real cases, among teetotalers, social drinkers, and heavy drinkers. A questionnaire, together with the informed consent form, was given to all the participants in order to evaluate alcohol intake in the one month before sample collection. Nail EtG levels in a social drinker were much higher than the concentrations of EtG in hair provided by the same subject, thus suggesting potential high sensitivity in evaluating both chronic excessive alcohol consumption and binge drinking habits.

Keywords: Ethyl glucuronide; Nails; Liquid chromatography tandem mass spectrometry; Alcohol markers; Binge drinking

Mn-doped ZnSe d-dots-based α-methylacyl-CoA racemase probe for human prostate cancer cell imaging by Xue Gao; Hao Zhang; Yang Li; Xingguang Su (pp. 1871-1877).

In this paper, we report the successful use of non-cadmium-based Mn-doped ZnSe d-dots (Mn/ZnSe) as highly efficient and nontoxic optical probes for human prostate cancer cells imaging. Mn/ZnSe d-dots are directly prepared in aqueous solution. The α-methylacyl-CoA racemase (AMACR) is overexpressed in prostate cancers; the presence of antibodies specific for AMACR is more sensitive and specific than serum prostate specific antigen levels in distinguishing patients with prostate cancers. Mn/ZnSe d-dots were linked to anti-AMACR to form Mn/ZnSe d-dots-anti-AMACR bioconjugates for the direct prostate cancer cell imaging. 3-(4,5-Dimethylthiazol-2-yl)-2 and 5-diphenyl tetrazolium bromide assay demonstrated that Mn/ZnSe d-dots exhibited favorable cytocompatibility to LNCaP cells with high concentration (1 mM) and long-time incubation (24 h). Furthermore, cellular imaging results demonstrated that Mn/ZnSe d-dots were remarkably efficacious for high-specificity cell imaging. The antibody-mediated delivery of the bioconjugates was further confirmed by the observation of no fluorescence signals in vitro targeting in nonprostate-cancer-based cell lines which are negative for AMACR. Mn/ZnSe d-dots as non-cadmium-based safe and efficient optical imaging nanoprobes could therefore be used for targeting imaging and treatment of cancers in the early stage.

Keywords: Mn/ZnSe d-dots; AMACR; Prostate cancer; Fluorescence imaging

Development of a routine method for the simultaneous confirmation and determination of clenbuterol in urine by minimal labeling isotope pattern deconvolution and GC-EI-MS by Ana González-Antuña; Pablo Rodríguez-González; Iván Lavandera; Giuseppe Centineo; Vicente Gotor; J. Ignacio García Alonso (pp. 1879-1888).

A novel and fast routine method for the simultaneous determination and confirmation of clenbuterol in bovine and human urine samples by gas chromatography electron ionization mass spectrometry (GC-EI-MS) has been developed. The method employs isotope dilution mass spectrometry (IDMS) and is based on a combination of minimal labeling (a single ¹³C label in the molecule) and isotope pattern deconvolution (IPD). This new methodology does not require the construction of a methodological calibration graph, and was compared with the classical IDMS procedure employed in clenbuterol analysis based on the use of a deuterated compound as internal standard (d₉-clenbuterol) and a calibration curve. The sample preparation consists of simple extraction with dichloromethane, which was dried and derivatized with chloro(chloromethyl)dimethylsilane, generating a cyclic dimethylsilamorpholine (DMS) derivative suitable for GC(EI)MS detection and identification. This compound produces five intense ions in the electron ionization source, which allow the presence of clenbuterol to be confirmed in just one analysis, as demanded by European Union directives. The accuracy of the method was studied by performing recovery experiments at different concentration levels (from 0.3 to 5 ng g⁻¹) in 5 mL bovine urine samples using two labeled compounds: an in-house-synthesized ¹³C₁-clenbuterol and a commercially available d₉-clenbuterol. The detection limit of the method in human urine was 0.050 ng g⁻¹ with a sample volume of 10 mL, and is thus suitable for antidoping control purposes. Finally, the ¹³C₁-clenbuterol standard was employed for the determination of clenbuterol in two reference materials, BCR-503 and BCR-504 (lyophilized bovine urine). The concentrations obtained were in agreement with the certified values, with a reproducibility of below 1% RSD. Figure A procedure for the determination and confirmation of clenbuterol in bovine and human urine samples by gas chromatography electron ionization mass spectrometry has been developed. In this method IDMS is employed in combination with minimal labelling and isotope pattern deconvolution.

Keywords: Clenbuterol; Minimal labeling; Isotope dilution; Food safety; Doping control

Two high-throughput screening assays for aberrant RNA–protein interactions in myotonic dystrophy type 1 by Catherine Z. Chen; Krzysztof Sobczak; Jason Hoskins; Noel Southall; Juan J. Marugan; Wei Zheng; Charles A. Thornton; Christopher P. Austin (pp. 1889-1898).

Myotonic dystrophy type 1 (DM1), the most prevalent form of adult muscular dystrophy, is caused by expansion of a CTG repeat in the 3′ untranslated region of the DM protein kinase (DMPK) gene. The pathogenic effects of the CTG expansion arise from the deleterious effects of the mutant transcript. RNA with expanded CUG tracts alters the activities of several RNA binding proteins, including muscleblind-like 1 (MBNL1). MBNL1 becomes sequestered in nuclear foci in complex with the expanded CUG-repeat RNA. The resulting loss of MBNL1 activity causes misregulated alternative splicing of multiple genes, leading to symptoms of DM1. The binding interaction between MBNL1 and mutant RNA could be a key step in the pathogenesis of DM1 and serves as a potential target for therapeutic intervention. We have developed two high-throughput screens suitable assays using both homogenous time-resolved fluorescence energy transfer and AlphaScreen technologies to detect the binding of a C-terminally His-tagged MBNL1 and a biotinylated (CUG)₁₂ RNA. These assays are homogenous and successfully miniaturized to 1,536-well plate format. Both assays were validated and show robust signal-to-basal ratios and Z′ factors.

Keywords: Myotonic dystrophy type 1; DM1; Muscleblind-like 1; MBNL1

Connecting simulated, bioanalytical, and molecular docking data on the stereoselective binding of (±)-catechin to human serum albumin by Myalowenkosy I. Sabela; Njabulo J. Gumede; Laura Escuder-Gilabert; Yolanda Martín-Biosca; Khirsna Bisetty; María-Jose Medina-Hernández; Salvador Sagrado (pp. 1899-1909).

The stereoselective binding of the frequently ingested nutraceutical (±)-catechin, with demonstrated differential biological activity between enantiomers, to human serum albumin (HSA), with the largest complexation and enantioselectivity potential among the plasmatic proteins, is studied by combining simulations to optimize the experimental design, robust in vitro electrokinetic chromatographic data, and molecular docking–chiral recognition estimates. Methodological and mathematical drawbacks in previous reports on (±)-catechin–HSA are detected and eliminated. Recent and novel direct equations extracted from the classical interaction model allows advantageous univariate mathematical data treatment, providing the first evidence of quantitative (±)-catechin–HSA enantioselectivity. Also, the binding site in HSA of the enantiomers is approached, and both the experimental enantioselectivity and the main binding site information are contrasted with a molecular docking approach.

Keywords: Stereoselective protein–ligand binding; Electrokinetic chromatography; Molecular docking–chiral recognition; Catechin; Human serum albumin

De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature by Jong-Seo Kim; Jin-Su Song; Yongju Kim; Seung Bum Park; Hie-Joon Kim (pp. 1911-1919).

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.

Keywords: De novo sequencing; N-terminal; Amidination; Bromine signature; MALDI signal enhancement

Identification of oligosaccharides from histopathological sections by MALDI imaging mass spectrometry by Masanori Yamada; Ikuko Yao; Takahiro Hayasaka; Masaru Ushijima; Masaaki Matsuura; Hideho Takada; Nobuaki Shikata; Mitsutoshi Setou; A-Hon Kwon; Seiji Ito (pp. 1921-1930).

Direct tissue analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) provides the means for in situ molecular analysis of a wide variety of biomolecules. This technology—known as imaging mass spectrometry (IMS)—allows the measurement of biomolecules in their native biological environments without the need for target-specific reagents such as antibodies. In this study, we applied the IMS technique to formalin-fixed paraffin-embedded samples to identify a substance(s) responsible for the intestinal obstruction caused by an unidentified foreign body. In advance of IMS analysis, some pretreatments were applied. After the deparaffinization of sections, samples were subjected to enzyme digestion. The sections co-crystallized with matrix were desorbed and ionized by a laser pulse with scanning. A combination of α-amylase digestion and the 2,5-dihydroxybenzoic acid matrix gave the best mass spectrum. With the IMS Convolution software which we developed, we could automatically extract meaningful signals from the IMS datasets. The representative peak values were m/z 1,013, 1,175, 1,337, 1,499, 1,661, 1,823, and 1,985. Thus, it was revealed that the material was polymer with a 162-Da unit size, calculated from the even intervals. In comparison with the mass spectra of the histopathological specimen and authentic materials, the main component coincided with amylopectin rather than amylose. Tandem MS analysis proved that the main components were oligosaccharides. Finally, we confirmed the identification of amylopectin by staining with periodic acid-Schiff and iodine. These results for the first time show the advantages of MALDI-IMS in combination with enzyme digestion for the direct analysis of oligosaccharides as a major component of histopathological samples.

Keywords: Imaging mass spectrometry; MALDI-TOF MS; Bioanalytical methods; Amylase; Amylopectin; Oligosaccharide

Development of a UHPLC-MS/MS method for the measurement of chlortetracycline degradation in swine manure by Weilin L. Shelver; Vincent H. Varel (pp. 1931-1939).

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed capable of simultaneously measuring chlortetracycline (CTC), epi-chlortetracycline, and isochlortetracycline (ICTC), as well as other structurally related tetracyclines in swine manure. A simple sample preparation was used consisting of extraction, dilution, centrifugation, and ultrafiltration. The concentrations of analyte were calculated using d₆-tetracycline as an internal standard in the matrix-matched standard curve. A solvent gradient resolved the compounds in 3.5 min with an additional 1.5 min of re-equilibration allowing the analyses of a large number of samples in a short period of time. MS/MS was used as the detection method giving analyte confirmation in addition to a large dynamic range and low detection limit. The UHPLC-MS/MS method successfully resolved multiple degradation products of CTC from the complex manure matrix. The method detection limits ranged from 1.9 pg/μL for CTC to 7.3 pg/μL for ICTC, and the calibration curve was linear from 1 to 10,000 pg/μL. The method was tested by measuring CTC and its degradation products as a function of time in incurred swine manure that had been incubated at three different temperatures (22 °C, 38 °C, and 55 °C). CTC concentration at 22 °C decreased 44% after 25 days; greater percentage decreases were observed when the manure was stored at elevated temperatures (96% and 98% for 38 °C and 55 °C, respectively). The concentration of the microbiologically inactivate isomer, ICTC, increased over the incubation period. At 22 °C, ICTC continued to increase through 25 days of incubation; at 38 °C, ICTC concentration plateaued on day 14 while at 55 °C ICTC concentration plateaued on day 7, with concentration increases of 198%, 374%, and 282% for 22 °C, 38 °C, and 55 °C, respectively. Figure Fate of chlortetracycline in swine manure digesters

Keywords: Analysis; Measurement; Manure treatment; Tandem MS

HS-SPME-GC×GC-qMS volatile metabolite profiling of Chrysolina herbacea frass and Mentha spp. leaves by Chiara Cordero; Simon Atsbaha Zebelo; Giorgio Gnavi; Alessandra Griglione; Carlo Bicchi; Massimo E. Maffei; Patrizia Rubiolo (pp. 1941-1952).

Headspace solid-phase microextraction (HS-SPME) comprehensive two-dimensional (2D) gas chromatography combined with quadrupole-mass spectrometry (GC×GC-qMS) with dedicated comparative data elaboration was applied to separate chemical patterns arising from the interaction between some Mentha species and the herbivore Chrysolina herbacea, also known as the mint bug. Upon feeding on different Mentha species (Mentha spicata L., Mentha × piperita L. and Mentha longifolia L.), C. herbacea produced frass (faeces) which were characterized by a typical volatile fraction. HS-SPME GC×GC-qMS analysis of the complex volatile fraction of both mint leaf and C. herbacea frass was submitted to advanced fingerprinting analysis of 2D chromatographic data. 1,8-Cineole, found in the leaves of all the Mentha species examined, was oxidized, and C. herbacea frass yielded high rates of several hydroxy-1,8-cineoles, including 2α-hydroxy-, 3α-hydroxy-, 3β-hydroxy- and 9-hydroxy-1,8-cineole. Upon insect feeding, several unknown oxidized monoterpenes, a p-menthane diol and three unknown phenylpropanoids were also detected in the frass volatiles. In M. longifolia, the occurrence of the monoterpene piperitenone oxide was found to be toxic and associated with insect death. The results of this work show that high throughput techniques such as HS-SPME and GC×GC-qMS fingerprint analysis are ideal tools to analyze complex volatile matrices, and provide a sensitive method for the direct comparison and chemical visualization of plant and insect emitted volatile components. Figure Because of feeding, Chrysolina. herbacea transformed terpenoids in M. spicata and M.x piperita and not in M. longifolia. Chrysolina herbacea frass, after feeding of the two species, contained oxidized 1,8-cineole derivatives namely 2α-hydroxy-, 3α-hydroxy-, 3β-hydroxy- and 9-hydroxy-1,8-cineole.

Keywords: Plant–insect interaction; Chrysolina herbacea ; Mentha spicata L.; Mentha × piperita L.; Mentha longifolia L.; Volatile fraction; HS-SPME-GC×GC-qMS; Fingerprint analysis

Determination of d-fagomine in buckwheat and mulberry by cation exchange HPLC/ESI–Q-MS by Susana Amézqueta; Esther Galán; Elisabet Fuguet; Montserrat Carrascal; Joaquín Abián; Josep Lluís Torres (pp. 1953-1960).

d-Fagomine is an iminosugar first found in buckwheat (Fagopyrum esculentum Moench) which if used as a dietary supplement or functional food component may reduce the risks of developing insulin resistance, becoming overweight and suffering from an excess of potentially pathogenic bacteria. As d-fagomine may become increasingly important to the food industry, a reliable analytical method for its determination in natural plant sources and foodstuffs is desirable. We have devised a method to separate d-fagomine from its diastereomers 3-epi-fagomine and 3,4-di-epi-fagomine in a single run by cation exchange high-performance liquid chromatography (HPLC) with detection and quantification by mass spectrometry using electrospray ionisation and a simple quadrupole analyser (ESI–Q-MS). The method is validated and applied to the determination of d-fagomine in buckwheat groats (6.7–44 mg kg⁻¹), leaves, bran and flour. We show that buckwheat contains 3,4-di-epi-fagomine (1.0–43 mg kg⁻¹), which has not previously been reported in this source. The procedure is also applied to mulberry (Morus alba) leaves, which contain d-fagomine and 3-epi-fagomine as minor components. The new method provides a means for convenient and accurate determination of d-fagomine in plant samples and foodstuffs. Figure Buckwheat groats contain d-fagomine and 3,4-di-epi-fagomine

Keywords: d-Fagomine; Iminosugars; Iminocyclitols; Buckwheat; Mulberry; HPLC/MS

Lactosylated casein phosphopeptides as specific indicators of heated milks by Gabriella Pinto; Simonetta Caira; Marina Cuollo; Olga Fierro; Maria Adalgisa Nicolai; Lina Chianese; Francesco Addeo (pp. 1961-1972).

Casein phosphopeptides (CPP) were identified in small amounts in milks heated at various intensities by using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. CPP selectively concentrated on hydroxyapatite (HA) were regenerated using phosphoric acid mixed in the matrix. Unphosphorylated peptides not retained by HA were removed by buffer washing. This procedure enhanced the MALDI signals of CPP that are ordinarily suppressed by the co-occurrence of unphosphorylated peptides. CPP, belonging to the β-casein (CN) family, i.e., (f1-29) 4P, (f1-28) 4P, and (f1-27) 4P, and the α_s2-CN family, i.e., (f1-21) 4P and (f1-24) 4P, were observed in liquid and powder milk. The lactosylated counterparts were specific to intensely heated milks, but absent in raw and thermized/pasteurized milk. Most CPP with C-terminal lysines probably arose from the activity of plasmin; an enzyme most active in casein hydrolysis. A CPP analogue was used as the internal standard. The raw milk signature peptide β-CN (f1-28) 4P constituted ~4.3% of the total β-CN. Small amounts of lactosylated peptides, which varied with heat treatment intensity, were detected in the milk samples. The limit of detection of ultra-high-temperature milk adjunction in raw or pasteurized milk was ~10%. Figure Schematic representation of the procedure for phosphoprotein/phosphopeptide enrichment using hydroxyapatite (HA). Native and lactosylated casein phosphopeptides are captured by HA, while non-phosphorylated peptide was washed out by the loading buffer. Signature peptides of UHT milk are detected through direct analysis by MALDI-TOF

Keywords: Hydroxyapatite-bound phosphopeptides (CPP) MALDI analysis; Lactosylated CPP detection; Signature CPP for heated milks

Collection of trace metals with cationic surfactant-silica particles followed by flotation with an anionic surfactant for seawater analysis by Hiroaki Matsumiya; Kengo Kitakata; Masataka Hiraide (pp. 1973-1977).

The analysis of seawater for trace metals is important for pollution monitoring and better understanding of marine systems. The present paper describes an efficient preconcentration method for the determination of trace metals in seawater. Trace metals [Ni(II), Cu(II), Ga(III), Cd(II), Pb(II), and Bi(III)] in 1,000 mL of seawater sample were complexed with ammonium pyrrolidinedithiocarbamate and sorbed onto silica particles covered with cetyltrimethylammonium chloride. After the addition of sodium dodecyl sulfate, the particles were floated to the solution surface by bubbling and then collected by suction. The trace metals were desorbed with dilute nitric acid and determined by inductively coupled plasma–mass spectrometry. The rapid 200-fold preconcentration was demonstrated with certified seawater samples.

Keywords: Flotation; Admicelle; Hemimicelle; Preconcentration; Seawater analysis; Inductively coupled plasma–mass spectrometry

Sections

Personal tools

Analytical and Bioanalytical Chemistry (v.402, #5)

Sections

Personal tools

Local resources

Analytical and Bioanalytical Chemistry (v.402, #5)