Analytica Chimica Acta (v.793, #C)

Display OmittedHuntington's disease (HD) is a neurodegenerative disorder caused by trinucleotide CAG (Cytosine–Adenine–Guanine) expansion on the Huntingtin gene (HTT) encoding for the Huntingtin protein (Htt). The protein has been linked in peripheral fibroblasts with dysregulation of cellular components which are part of lipid rafts in plasma membrane sub-domains. Therefore the analysis of the plasma membrane might be a useful diagnostic biomarker for the detection of the presence and possible onset of HD in readily accessible peripheral cells. Here Raman spectroscopy has been used with a chemometric approach in the form of Partial Least Square (PLS) for an initial corroboration that the plasma membrane is indeed a sub-cellular biomarker discriminator for HD identification. Observations were made in the spectral regions from 400 to 1800 cm−1 and 2700 to 3200 cm−1 with the former region displaying the most significant differences and peak displacement between plasma membranes extracted from HD and control fibroblast cells. The major differences in plasma membrane composition reside in sub-cellular elements putatively associated to cholesterol, phospholipids (mainly phophatidylinositol) as well as proteins containing tyrosine. These findings are indicative of the plasma membrane as an amenable biomarker for HD for further in vitro research with possible applications in vivo models.
Keywords: Raman spectroscopy; Partial least square (PLS); Chemometrics; Huntington's disease; Plasma membrane; Biomarker;

Fabrication and characterisation of high performance polypyrrole modified microarray sensor for ascorbic acid determination by J. Samseya; R. Srinivasan; Yu-Tsern Chang; Cheng-Wen Tsao; V.S. Vasantha (11-18).
In this study, gold microelectrode array (Au/MEA) with electrode of 12 μm diameter was fabricated by photolithography technique. Subsequently, polypyrrole (Ppy) modified gold microarrays sensor (Ppy/Au/MEA) was prepared by cyclic voltammetry technique. The deposition potential range and number of cycles were optimised in order to get optimum thickness of Ppy film. Scanning Electron Microscope and Atomic Force Microscope investigations reveal that Ppy coating formed at 3 cycles is porous with thickness of 1.5 μm which exhibiting high catalytic current for ascorbic acid (AA) in square wave technique (SWV). In contrast to earlier sensors designs, these Ppy/Au/MEA sensors exhibits lower detection limit (LOD) of 10 nm towards AA at physiological conditions. It also exhibits enhanced sensitivity (2.5 mA cm−2  mM−1) and long range of linear detection limit from 10 nm to 2.8 mM. In the same way, polypyrrole modified macro Au (Ppy/Au/MA) biosensor was also fabricated and its electro catalytic property towards AA was compared with that of Ppy/Au/MEA. The Ppy/Au/MA exhibits sensitivity of only 0.27 mA cm−2  mM−1, LOD of 5 μM and linear range of 10 μM to 2.2 mM. Hence, our investigations indicate that the Ppy/Au/MEA could serve as highly sensitive sensor for AA than any of the earlier designs. So, the Ppy/Au/MEA electrode was utilised for determination AA in a wide variety of real samples.
Keywords: Gold micro array electrode; Ascorbic acid; Cyclic voltammetry; Square wave voltammetry; Polypyrrole;

A new kind of signal amplification strategy based on ferrocene (Fc) incorporated polystyrene spheres (PS-Fc) was proposed. The synthesized PS-Fc displayed narrow size distribution and good stability. PS-Fc was applied as label to develop immunosensors for prostate specific antigen (PSA) after the typical sandwich immunoreaction by linking anti-PSA antibody (Ab2) onto PS-Fc. After the fabrication of the immunosensor, tetrahydrofuran (THF) was dropped to dissolve PS and release the contained Fc for the following stripping voltammetric detection. PS-Fc as a new electrochemical label prevented the leakage of Fc and greatly amplified the immunosensor signal. In addition, the good biocompatibility of PS could maintain the bioactivity of the antibodies. The response current was linear to the logarithm of PSA concentration in the range from 0.01 ng mL−1 to 20 ng mL−1 with a detection limit of 1 pg mL−1. The immunosensor results were validated through the detection of PSA in serum samples with satisfactory results.
Keywords: Immunosensor; Polystyrene sphere; Ferrocene; Graphene oxide; Prostate specific antigen;

Display OmittedA gas chromatographic–positive chemical ionisation-tandem mass spectrometric (GC–PCI-MS/MS) method for the simultaneous determination of 10 oxidative metabolites of the monoterpenoid hydrocarbons α-pinene, (R)-limonene, and Δ3-carene ((+)-3-carene) in human urine was developed and tested for the monoterpene biomonitoring of the general population (n  = 36). The method involves enzymatic cleavage of the glucuronides followed by solid-supported liquid–liquid extraction and derivatisation using a two-step reaction with N,O-bis(trimethylsilyl)-trifluoroacetamide and N-(trimethylsilyl)imidazole. The method proved to be both sensitive and reliable with detection limits ranging from 0.1 to 0.3 μg L−1. In contrast to the frequent and distinct quantities of (1S,2S,4R)-limonene-1,2-diol, the (1R,2R,4R)-stereoisomer could not be detected. The expected metabolite of (+)-3-carene, 3-caren-10-ol was not detected in any of the samples. All other metabolites were detected in almost all urine samples.The procedure enables for the first time the analysis of trace levels of a broad spectrum of mono- and bicyclic monoterpenoid metabolites (alcohols, diols, and carboxylic acids) in human urine. This analytical procedure is a powerful tool for population studies as well as for the discovery of human metabolism and toxicokinetics of monoterpenes.
Keywords: Biomonitoring; (R)-Limonene; α-Pinene; Δ3-Carene; Urine; Human metabolites;

Low-density magnetofluid dispersive liquid–liquid microextraction for the fast determination of organochlorine pesticides in water samples by GC-ECD by Zhigang Shen; Zeying He; Peng Wang; Zhiqiang Zhou; Mingjing Sun; Jingdong Li; Donghui Liu (37-43).
A new micro-extraction technique named low-density magnetofluid dispersive liquid–liquid microextraction (LMF-DMMLE) has been developed, which permits a wider range of solvents and can be combined with various detection methods. Comparing with the existing low density solvents micro-extraction methods, no special devices and complicated operations were required during the whole extraction process. Dispersion of the low-density magnetofluid into the aqueous sample is achieved by using vortex mixing, so disperser solvent was unnecessary. The extraction solvent was collected conveniently with an external magnetic field placed outside the extraction container after dispersing. Then, the magnetic nanoparticles were easily removed by adding precipitation reagent under the magnetic field. In order to evaluate the validity of this method, ten organochlorine pesticides (OCPs) were chosen as the analytes. Parameters influencing the extraction efficiency such as extraction solvents, volume of extraction solvents, extraction time, and ionic strength were investigated and optimized. Under the optimized conditions, this method showed high extraction efficiency with low limits of detection of 1.8–8.4 ng L−1, good linearity in the range of 0.05–10.00 μg L−1 and the precisions were in the range of 1.3–9.6% (RSD, n  = 5). Finally, this method was successfully applied in the determination of OCPs in real water samples.
Keywords: Dispersive magnetic liquid micro-extraction; Low-density solvent; Organochlorine pesticides; GC-ECD; Water;

‘Metal-free’ seawater doped with varying concentrations of ‘zero’ isotope standards, processed through our simultaneous method, and then analyzed by double spike MC-ICPMS for Fe, Zn and Cd isotope ratios. All values were determined within 2 σ error (error bars shown) of zero.The study of Fe, Zn and Cd stable isotopes (δ56Fe, δ66Zn and δ114Cd) in seawater is a new field, which promises to elucidate the marine cycling of these bioactive trace metals. However, the analytical challenges posed by the low concentration of these metals in seawater has meant that previous studies have typically required large sample volumes, highly limiting data collection in the oceans. Here, we present the first simultaneous method for the determination of these three isotope systems in seawater, using Nobias PA-1 chelating resin to extract metals from seawater, purification by anion exchange chromatography, and analysis by double spike MC-ICPMS. This method is designed for use on only a single litre of seawater and has blanks of 0.3, 0.06 and <0.03 ng for Fe, Zn and Cd respectively, representing a 1–20 fold reduction in sample size and a 4–130 decrease in blank compared to previously reported methods. The procedure yields data with high precision for all three elements (typically 0.02–0.2‰; 1σ internal precision), allowing us to distinguish natural variability in the oceans, which spans 1–3‰ for all three isotope systems. Simultaneous extraction and purification of three metals makes this method ideal for high-resolution, large-scale endeavours such as the GEOTRACES program.
Keywords: Iron; Zinc; Cadmium; Isotopic; Marine; Nobias;

Superoxide anion radical (O2) plays an important role in several human diseases. The xanthine/xanthine oxidase system is frequently utilized to produce O2. However, false positive results are easily got by using this system. The common spectrophotometric probes for O2 are nitroblue tetrazolium (NBT) and cytochrome c. Nevertheless, the application of NBT method is limited because of the water-insolubility of NBT formazan and the assay using cytochrome c lacks sensitivity and is not suitable for microplate measurement. We overcome these problems by using 1,2,3-trihydroxybenzene (pyrogallol) as O2-generating system and a highly water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-1) which can be reduced by superoxide anion radical to a stable water-soluble formazan with a high absorbance at 450 nm. The method is simple, rapid and sensitive. Moreover, it can be adapted to microplate format. In this study, the O2 scavenging activities of superoxide dismutase (SOD), l-ascorbic acid, N-acetyl-l-cysteine (NAC), albumin from human serum, flavonoids and herbal extracts were assessed by using this method. Meanwhile, the activities of tissue homogenates and serum were determined by using this validated method. This method, applicable to tissue homogenates, serum and herbal extracts, proved to be efficient for measuring O2 scavenging activities of biological and abiological samples.
Keywords: Superoxide anion radical; Pyrogallol; WST-1; O2 scavenging activity;

Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry by Sabina Strano-Rossi; Erika Castrignanò; Luca Anzillotti; Sara Odoardi; Fabio De-Giorgio; Ana Bermejo; Vincenzo L. Pascali (61-71).
A method for the screening of various anabolic steroids and their esters in human hair, based on liquid-chromatography–high resolution mass spectrometry using an Exactive benchtop Orbitrap mass spectrometer, has been set up and validated. This method involved methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a C18 column. The mass detector, with nominal resolving power of 100,000, operated in full scan mode in APCI under positive ionization mode. Analytes were identified by exact mass, correspondence of isotopic cluster and retention times.The limits of detection obtained varied from 10 to 50 pg mg−1, and limits of quantitation were 0.5 ng mg−1 for all compounds. The method was linear for all analytes in the ranges from the LOQ to 6 ng mg−1, giving correlation coefficients >0.99 for all analytes. Also accuracy (intended as %E) and repeatability (%CV) were always lower than 15%. Specificity was assessed by analysing ten blank samples and fifteen samples from polidrug abusers. This method was applied to a real-life case, resulting in the identification of testosterone undecanoate in the hair of a suspect. The analyte identity was confirmed by the analysis of its in-source fragmentation and comparison to a certified standard. Thanks to the scan acquisition, this method also enables retrospective re-analysis of the acquired datafile in case a further analyte needs to be screened.
Keywords: Anabolic steroids; Esters; Hair analysis; High resolution mass spectrometry; Orbitrap; Forensic toxicology;

Elemental analyses of soil and sediment fused with lithium borate using isotope dilution laser ablation-inductively coupled plasma-mass spectrometry by Julien Malherbe; Fanny Claverie; Aitor Alvarez; Beatriz Fernandez; Rosario Pereiro; John L. Molloy (72-78).
Quantitative analysis using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) remains challenging primarily due to the lack of appropriate reference materials available for the wide variety of samples of interest and to elemental fractionation effects. Isotopic dilution mass spectrometry (IDMS) is becoming the methodology of choice to address these issues because the different isotopes of an element represent near-perfect internal standards. In this work, we investigated the lithium borate fusion of powdered solid samples, including soils, sediments, rock mine waste and a meteorite, as a strategy to homogenously distribute, i.e. equilibrate the elements and the added isotopically enriched standards. A comparison of this methodology using two pulsed laser ablation systems (ArF* excimer and Nd:YAG) with different wavelengths as well as two ICP-MS instruments (quadrupole and double-focusing sector field) was performed. Emphasis was put on using standard equipment to show the potential of the proposed strategy for its application in routine laboratories. Cr, Zn, Ba, Sr and Pb were successfully determined by LA-ICP-IDMS in six Standard Reference Materials (SRMs) representing different matrices of environmental interest. Experimental results showed the SRM fused glasses exhibited a low level of heterogeneity (intra- and inter-sample) for both natural abundance and isotopically enriched samples (RSD <3%, n  = 3, 1σ). A good agreement between experimental results and the certified values was also observed.
Keywords: Laser ablation inductively coupled plasma mass spectrometry; Trace element; Isotope dilution mass spectrometry; Geological reference materials;

N-acetylcysteine induced quenching of red fluorescent oligonucleotide-stabilized silver nanoclusters and the application in pharmaceutical detection by Xinyi Wang; Ruoyun Lin; Zhihan Xu; Hongduan Huang; Limei Li; Feng Liu; Na Li; Xiaoda Yang (79-85).
In this work, we reported a new, simple and sensitive method for determination of N-acetylcysteine (NAC) based on quenching of the red fluorescence of oligonuleotide-protected silver nanoculsters (Ag NCs) with the quantum yield of 68.3 ± 0.3%. This method was successfully used for the assay of NAC granules presenting a linear range from 100 nM to 1200 nM (LOD of 50 nM) with minimal interferences from potential coexisting substances. It is for the first time that quenching performance of the thiol-containing compound was found to follow a non-linear Stern–Volmer profile, indicative of a complicated quenching mechanism with static quenching dominating, in which DNA-template of Ag NCs was partly replaced by NAC, as elucidated by spectral investigations. This study extended the analytical application of silver nanoclusters as well as provided a more insightful understanding of the quenching mechanism of thiol-compounds on the fluorescence of Ag NCs.
Keywords: Fluorescence; Quenching; Silver nanoclusters; Nucleotide; N-acetylcysteine;

A label-free and signal-on supersandwich fluorescent platform for Hg2+ sensing by Tao Yuan; Lianzhe Hu; Zhongyuan Liu; Wenjing Qi; Shuyun Zhu; Aziz-ur-Rehman; Guobao Xu (86-89).
A novel label-free supersandwich fluorescent detection platform has been demonstrated for the first time. The platform allows sensitive, selective and rapid detection of Hg2+.A label-free supersandwich fluorescent assay was demonstrated for the first time by taking Hg2+ as a detection candidate. The principle of the proposed supersandwich fluorescent platform is based on the formation of supersandwich structure by T-Hg2+-T coordination and the fluorescence enhancement of the intercalated Genefinder (GF) in double strand DNA (dsDNA). Such supersandwich fluorescent DNA sensor exhibits a linear range of 10–300 nM for the detection of Hg2+, with a detection limit of 2.5 nM on the basis of the 3σ/slope (σ represents the standard deviation of the blank samples), which is well below the permit of the U.S. Environmental Protection Agency (<10 nM). The detection can be fulfilled in less than 10 min. The proposed mix-and-detect fluorescent platform exhibits excellent sensitivity, selectivity, and convenient manipulation. The assay was successfully used to detect Hg2+ in the lake water samples, which suggested its potential in practical samples.
Keywords: Fluorescence; Label-free; Supersandwich; Signal-on; Hg2+;

This paper reports the development of a facile and effective approach, based on the use of Zr-based metal-organic frameworks (UiO-66) sensor with micropores geometry, shape and particle morphology for the visual detection and removal of ultra-traces of some toxic metal ions such as Bi(III), Zn(II), Pb(II), Hg(II) and Cd(II). UiO-66 was used as selective carriers for accommodating hydrophobic chromophore probes such as dithizone (DZ) without coupling agent for sensitive and selective discrimination of trace level of toxic analytes. The developed UiO-66 sensor was utilized for the detection of ultra-traces of some toxic metal ions with the naked eye. The new sensor displays high sensitivity and selectivity of a wide range of detectable metals analytes up to 10−10  mol dm−3 in solution, in a rapid analyte uptake response (seconds). The developed sensor is stable, cost effective, easy to prepare, and would be useful for rapid detection and removal of ultra-traces of toxic metal ions in water samples.
Keywords: UiO-66; Toxic metals; Chemosensor; Optical sensor; Discrimination;

Aggregates of hexaphenylbenzene derivatives 3, having pyrene groups form network of fluorescent nanofibres in presence of mercury ions. Further, fluorescent nanofibres of 3-Hg 2+ supramolecular ensemble exhibit sensitive and pronounced response towards the picric acid. In addition, the solution coated paper strips of 3-Hg 2+ supramolecular ensemble can detect picric acid in the range of 2.29 fg/cm2, thus, providing a simple, portable and low cost method for detection of picric acid in solution and in contact mode.Aggregates of hexaphenylbenzene derivatives 3, having pyrene groups form network of fluorescent nanofibres in presence of mercury ions. Further, fluorescent nanofibres of 3-Hg 2+ supramolecular ensemble exhibit sensitive and pronounced response towards the picric acid. In addition, the solution coated paper strips of 3-Hg 2+ supramolecular ensemble can detect picric acid in the range of 2.29 fg/cm2, thus, providing a simple, portable and low cost method for detection of picric acid in solution and in contact mode.
Keywords: Hexaphenylbenzene; AIEE; Hg2+ chemosensor; Self assembled nanofibres; Femtogram level detection of PA;

At present, the analytical method for paralytic shellfish poisoning (PSP) toxins in shellfish is the mouse bioassay (MBA), which is an official method of the Association of Analytical Communities (AOAC [8]). However, the low sensitivity and concerns over the number of live animals required for testing have been cited as the major reason for seeking its replacement. In this report, we employed an open-sandwich immunoassay (OS-IA) to detect gonyautoxin (GTX2/3), a kind of PSP toxins. OS-IA, which utilizes the antigen-induced enhancement of antibody VH/VL interaction, can measure a small molecule antigen in a noncompetitive format. Hence it has a wider working range and shorter measurement time. We isolated anti-GTX2/3 antibody gene from a hybridoma GT-13A by screening a Fab-displaying phage library. Then the vectors for OS-IA were constructed, and examined for antigen concentration-dependency of the VH/VL interaction by OS-ELISA. As a result, in each case, signal intensity increases notably in a wide concentration range (0.1 to >1000 ng mL−1) of free GTX2/3, which was enough to cover its regulation value (80 μg 100 g−1) in many countries. So OS-IA will be widely applicable to detect PSP toxins in shellfish meats and in drinking water.
Keywords: Open sandwich immunoassay; Gonyautoxin; PSP toxins; Phage display; Maltose binding protein; Hapten;

Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis is a prominent bioseparation method based on the mobility diversity caused by sequence-induced conformational differences of single-stranded DNA. The use of Pluronic polymer matrix has opened up new opportunities for CE-SSCP, because it improved the resolution for various genetic analyses. However, there still exists a challenge in optimizing Pluronic-based CE-SSCP, because the physical properties of Pluronic solutions are sensitive to temperature, particularly near the gelation temperature, where the viscoelasticity of Pluronic F108 solutions sharply changes from that of a Newtonian fluid to a hydrogel upon heating. We have focused on a set of experiments to control the ambient temperature of the CE system with the aim of enhancing the reliability of the CE-SSCP analysis by using the Applied Biosystems ABI 3130xl genetic analyzer with Pluronic F108 solution matrix. The ambient temperature control allowed us to vary the inlet and outlet portion of the capillary column, while the temperature of the column was kept at 35 °C. The resolution to separate 2 single-base-pair-differing DNA fragments was significantly enhanced by changing the temperature from 19 to 30 °C. The viscoelastic properties of the F108 solution matrix upon heating were also investigated by ex situ rheological experiments with an effort to reveal how the development of gels in Pluronic solutions affects the resolution of CE-SSCP. We found that the column inlet and outlet temperatures of the capillary column have to be controlled to optimize the resolution in CE-SSCP by using the Pluronic matrix.
Keywords: Capillary electrophoresis-single strand conformation polymorphism; High resolution; Ambient temperature; Polymer matrix;