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Analytica Chimica Acta (v.771, #)
A new approach to polarimetric measurements based on birefringent crystals and diode lasers by Lívia Paulia Dias Ribeiro; Jarbas José Rodrigues Rohwedder; Celio Pasquini (pp. 1-6).
Display Omitted► New approach to polarimetric measurements is evaluated. ► A robust, with no mechanical moving parts polarimeter is presented. ► The performance of the instrument was evaluated for saccharimetric measurements. ► The uncertainty of the instrument was evaluated as a function of the measured angle. ► Polarimeter allow the use of low cost lasers while obtaining precision as good as 0.003°.A new polarimetric instrument and measurement method is described based on the use of diode lasers as radiation source (532, 650 and 1064nm) and birefringent prisms, such as Glan-Laser and Wollaston, as analyzers. The laser radiation is passed through a dichroic polarizer film for further orientation of its polarization plane at 45° in relation to the polarization plane of the analyzer. The polarized beam, oriented in that way, passes the sample cell, impinges the prism surface, and the intensities of the two emerged beams are detected by two twin silicon detectors. Ideally, in the absence of any optically active substances, the crystals produces two orthogonally polarized refracted beams of equal intensity. In the presence of an optically active substance, the arctangent of the square root of the beam intensities ratio is equal to the new polarization angle ( β) of the laser beam. The rotation angle imposed for any optically active substance present in the sample cell is then given by: α=(45– β)°. Because the rotation is obtained by the ratio of the intensities of two beams, it is independent of the laser intensity, which can vary up to ±15% with no significant effect on the accuracy of the polarimetric measurement. The instrument has been evaluated for measurement of optically active substances such as sucrose and fructose. The instrument employs low cost components, is capable of attaining a repeatability of ±0.003° and can measure the rotation angle, over a ±45° range, in less than 2s. Because it does not present any moving parts it can be easily adapted for in/on-line process monitoring of optically active substances.
Keywords: Polarimetry; Birefringent prisms; Polarimeter; Optically active substances
Mixture models for two-dimensional baseline correction, applied to artifact elimination in time-resolved spectroscopy by Johan J. de Rooi; Olivier Devos; Michel Sliwa; Cyril Ruckebusch; Paul H.C. Eilers (pp. 7-13).
Display Omitted► Penalized regression with P-splines to estimate a two-dimensional surface. ► For images and applications where anisotropic smoothing is required. ► Provides powerful baseline correction procedure for two-dimensional data. ► To correct for coherent artifact signals in ultrafast time-resolved spectra.Baseline correction and artifact removal are important pre-processing steps in analytical chemistry. We propose a correction algorithm using a mixture model in combination with penalized regression. The model is an extension of a method recently introduced for baseline estimation in the case of one-dimensional data. The data are modeled as a smooth surface using tensor product P-splines. The weights of the P-splines regression model are computed from a mixture model where a datapoint is either allocated to the noise around the baseline, or to the artifact component. The method is broadly applicable for anisotropic smoothing of two-way data such as two-dimensional gel electrophoresis and two-dimensional chromatography data. We focus here on the application of the approach in femtosecond time-resolved spectroscopy, to eliminate strong artifact signals from the solvent.
Keywords: Baseline estimation; P-splines; Tensor product; Mixture model; Two-dimensional data; Time-resolved spectroscopy
Simultaneous determination of uric acid, xanthine, hypoxanthine and caffeine in human blood serum and urine samples using electrochemically reduced graphene oxide modified electrode by M. Amal Raj; S. Abraham John (pp. 14-20).
Display Omitted► Glassy carbon electrode was modified with graphene by self assembly method. ► Modified electrode was characterized by Raman spectroscopy. ► Graphene modified electrode separated the voltammetric signals of four purine derivatives. ► Selective and simultaneous determination of four purine compounds were achieved. ► Practical application was demonstrated in human blood serum and urine samples.This paper describes the fabrication of graphene on glassy carbon electrode (GCE) by electrochemical reduction of graphene oxide (GO) attached through 1,6-hexadiamine on GCE and the simultaneous determination of structurally similar four purine derivatives using the resultant electrochemically reduced GO (ERGO) modified electrode. The electrocatalytic activity of ERGO was investigated toward the oxidation of four important purine derivatives, uric acid (UA), xanthine (XN), hypoxanthine (HXN) and caffeine (CAF) at physiological pH. The modified electrode not only enhanced the oxidation currents of the four purine derivatives but also shifted their oxidation potentials toward less positive potentials in contrast to bare GCE. Further, it successfully separates the voltammetric signals of the four purine derivatives in a mixture and hence used for the simultaneous determination of them. Selective determination of one purine derivative in the presence of low concentrations other three purine derivatives was also realized at the present modified electrode. Using differential pulse voltammetry, detection limits of 8.8×10−8M, 1.1×10−7M, 3.2×10−7M and 4.3×10−7M were obtained for UA, XN, HXN and CAF, respectively. The practical application of the modified electrode was demonstrated by simultaneously determining the concentrations of UA, XN, HXN and CAF in human blood plasma and urine samples.
Keywords: Graphene oxide; Electrochemical reduction; Raman spectra; Purine derivatives; Human blood plasma
Fabrication and application of a new modified electrochemical sensor using nano-silica and a newly synthesized Schiff base for simultaneous determination of Cd2+, Cu2+ and Hg2+ ions in water and some foodstuff samples by Abbas Afkhami; Farzaneh Soltani-Felehgari; Tayyebeh Madrakian; Hamed Ghaedi; Majid Rezaeivala (pp. 21-30).
Display Omitted► A new modified electrochemical sensor was constructed and used. ► A new Schiff base coated nano-silica was used as modifier. ► The electrochemical properties of electrode were studied. ► This modifier enhanced the electrochemical properties of electrode. ► The electrode was used for simultaneous determination of Cd2+, Cu2+ and Hg2+ ions.A new chemically modified carbon paste electrode was constructed and used for rapid, simple, accurate, selective and highly sensitive simultaneous determination of cadmium, copper and mercury using square wave anodic stripping voltammetry (SWASV). The carbon paste electrode was modified by N,N′-bis(3-(2-thenylidenimino)propyl)piperazine coated silica nanoparticles. Compared with carbon paste electrode, the stripping peak currents had a significant increase at the modified electrode. Under the optimized conditions (deposition potential, −1.100V vs. Ag/AgCl; deposition time, 60s; resting time, 10s; SW frequency, 25Hz; pulse amplitude, 0.15V; dc voltage step height, 4.4mV), the detection limit was 0.3, 0.1 and 0.05ngmL−1 for the determination of Cd2+, Cu2+ and Hg2+, respectively. The complexation reaction of the ligand with several metal cations in methanol was studied and the stability constants of the complexes were obtained. The effects of different cations and anions on the simultaneous determination of metal ions were studied and it was found that the electrode is highly selective for the simultaneous determination of Cd2+, Cu2+ and Hg2+. Furthermore, the present method was applied to the determination of Cd2+, Cu2+ and Hg2+ in water and some foodstuff samples.
Keywords: Simultaneous determination; Cd; 2+; , Cu; 2+; and Hg; 2+; determination; Chemically modified electrodes, Square wave stripping voltammetry; Foodstuff samples; Water samples
N-Methylimidazolium modified magnetic particles as adsorbents for solid phase extraction of genomic deoxyribonucleic acid from genetically modified soybeans by Manchen Deng; Cheng Jiang; Li Jia (pp. 31-36).
Display Omitted► The MIm-MPs based MSPE method was developed for extraction of DNA from GM soybeans. ► The method does not use harmful reagents. ► High yield and high quality of DNA are obtained. N-Methylimidazolium modified magnetic particles (MIm-MPs) were prepared and applied in the solid phase extraction of genomic deoxyribonucleic acid (DNA) from genetically modified soybeans. The adsorption of MIm-MPs for DNA mainly resulted from the strong electrostatic interaction between the positively charged MPs and the negatively charged DNA. The elution of DNA from MPs–DNA conjugates using phosphate buffer resulted from the stronger electrostatic interaction of phosphate ions with MPs than DNA. In the extraction procedure, no harmful reagents (e.g. phenol, chloroform and isopropanol, etc.) used, high yield (10.4μg DNA per 30mg sample) and high quality ( A260/ A280=1.82) of DNA can be realized. The as-prepared DNA was used as template for duplex-polymerase chain reaction (PCR) and the PCR products were analyzed by a sieving capillary electrophoresis method. Quick and high quality extraction of DNA template, and fast and high resolution detection of duplex PCR products can be realized using the developed method. No toxic reagents are used throughout the method.
Keywords: N; -Methylimidazolium; Magnetic solid phase extraction; Deoxyribonucleic acid; Capillary electrophoresis; Polymerase chain reaction; Genetically modified soybeans
Application of magnetic and core–shell nanoparticles to determine enrofloxacin and its metabolite using laser induced fluorescence microscope by Suji Kim; Junga Ko; H.B. Lim (pp. 37-41).
Laser induced fluorescence microscopy and core–shell nanoparticles to determine antibiotics.Display Omitted► An analytical method using two different nanoparticles to determine antibiotics. ► Dye-doped core–shell silica nanoparticles were used for fluorescent tagging while magnetic particles were used for concentrating the enrofloxacin. ► Improvement of the detection limit about 54-fold compared to ELISA.A unique analytical method using nanoparticles and laser-induced fluorescence microscopy (LIFM) was developed to determine enrofloxacin in this work. For sample pretreatment, two different kinds of particles, i.e., synthesized dye-doped core–shell silica nanoparticles and magnetic micro-particles (MPs), were used for fluorescent tagging and concentrating the enrofloxacin, respectively. The antibody of enrofloxacin was immobilized on the synthesized FITC-doped core–shell nanoparticles, and the enrofloxacin target was extracted by the MPs. At this moment, the average number of antibodies on each core–shell silica nanoparticle was ∼0.9, which was determined by the fluorescence ratiometric method. The described method was demonstrated for a meat sample to determine enrofloxacin using LIFM, and the result was compared with enzyme-linked immunosorbent assay (ELISA). The developed technique allowed the simplified analytical procedure, improved the detection limit about 54-fold compared to ELISA.
Keywords: Abbreviations; MP; magnetic micro-particles; NP; nanoparticle; SNP; silica nanoparticle; LIFM; laser induced fluorescence microscope; HPLC; high pressure liquid chromatography; LC–MS; liquid chromatography–mass spectrometry; ELISA; enzyme-linked immunosorbent assay; STUN; sample treatment using nanoparticles; SEM; scanning electron microscopy; PMT; photomultiplier tube; CCD; charge-coupled device; DPSS laser; diode-pumped solid-state laser; FITC; fluorescein isothiocyanate; Cy5; cyanine dye (5); EDC; N; -(3-dimethylaminopropyl)-; N′; -ethylcarbodiimide; NHS; N; -hydroxysuccinimide; PBS; phosphate-buffered salineDye-doped core–shell silica nanoparticles; Magnetic nanoparticles; Laser-induced fluorescence; Determination of enrofloxacin; Antibiotics; Sample treatment using nanoparticles
Validation of the analytical procedure for the determination of the neurotoxin β-N-methylamino-l-alanine in complex environmental samples by Audrey Combes; Saïda El Abdellaoui; Cédric Sarazin; Jérome Vial; Annick Mejean; Olivier Ploux; Valérie Pichon (pp. 42-49).
Display Omitted► LC/MS–MS quantification ofl-BMAA at trace levels. ► Limit of quantification of 2ngmL−1 in freshwater and 0.6ngmg−1 in cyanobacteria. ► Monitoring of the global yield of the analytical method using an internal standard. ► Validation of the method by the total error approach.The neurotoxicl-2-amino-3-methylaminopropionic acid (BMAA) was hypothesized to be involved in sporadic cases of amyotrophic lateral sclerosis (ALS). Studies highlighting a possible implication of environmental factors in the incidence of sporadic ALS have become more numerous over recent years. Over the past years, the most widely used method for quantifying BMAA was based on the derivatization of this polar and basic molecule with a fluorescent compound (6-aminoquinolonyl-N-hydroxysuccinimidyl, 6-AQC). This derivatization allows the retention of the conjugate by reversed-phase liquid chromatography and its detection by fluorescence. Nevertheless, recent findings have shown that this method applied to complex samples may cause false positive responses. We therefore developed an analytical procedure for the determination of underivatized BMAA at trace level in complex environmental matrices (river water, cyanobacteria and biofilm) using solid-phase extraction (SPE) based on mixed mode sorbent to concentrate and clean up real samples. Analyzes were performed by hydrophilic interaction chromatography (HILIC) coupled to electrospray ionization and tandem mass spectrometry used in multiple reaction monitoring scan mode. Analytical procedures were validated for the different natural samples using the total error approach. BMAA can be quantified by these reliable and highly selective analytical methods in a range of only a few ngmL−1 in river water and a few ngmg−1 dry weight in cyanobacteria and biofilm matrices.
Keywords: β-N-methylamino-; l; -alanine; Total error approach; Cyanobacteria; Freshwater; Solid-phase extraction
On-line liquid phase micro-extraction based on drop-in-plug sequential injection lab-at-valve platform for metal determination by Constantina Mitani; Aristidis N. Anthemidis (pp. 50-55).
Display Omitted► Drop-in-plug micro-extraction based on SI-LAV platform for metal preconcentration. ► Automatic liquid phase micro-extraction coupled with FAAS. ► Organic solvents with density higher than water are used. ► Lead determination in environmental water and urine samples.A novel automatic on-line liquid phase micro-extraction method based on drop-in-plug sequential injection lab-at-valve (LAV) platform was proposed for metal preconcentration and determination. A flow-through micro-extraction chamber mounted at the selection valve was adopted without the need of sophisticated lab-on-valve components. Coupled to flame atomic absorption spectrometry (FAAS), the potential of this lab-at-valve scheme is demonstrated for trace lead determination in environmental and biological water samples. A hydrophobic complex of lead with ammonium pyrrolidine dithiocarbamate (APDC) was formed on-line and subsequently extracted into an 80μL plug of chloroform. The extraction procedure was performed by forming micro-droplets of aqueous phase into the plug of the extractant. All critical parameters that affect the efficiency of the system were studied and optimized. The proposed method offered good performance characteristics and high preconcentration ratios. For 10mL sample consumption an enhancement factor of 125 was obtained. The detection limit was 1.8μgL−1 and the precision expressed as relative standard deviation (RSD) at 50.0μgL−1 of lead was 2.9%. The proposed method was evaluated by analyzing certified reference materials and applied for lead determination in natural waters and urine samples.
Keywords: Sequential injection; Lab-at-valve; Drop-in-plug micro-extraction; Atomic spectrometry; Lead determination
Improvement using chemometrics in ion mobility coupled to mass spectrometry as a tool for mass spectrometry fragmentation studies: Flavonoid aglycone cases by S. Garmón-Lobato; B. Abad-García; M.B. Sánchez-Ilárduya; M. Romera-Fernández; L.A. Berrueta; B. Gallo; F. Vicente (pp. 56-64).
Display Omitted► We propose to use IMS coupled to MS for study fragmentations, and relationships between precursor ions and product ions. ► Coshift alignment and CLS fitting is used to improve IMS selectivity and accuracy. ► Proposed method is successfully applied for studying flavonoid agycones. ► Proposed method is automatable and its information is similar to a full set of MS3 experiments.A chemometric method for the treatment of ion mobility coupled to mass spectrometry (IMS/MS) data is proposed as a complementary tool for obtaining experimental evidence for the study of MS fragmentations, which can provide a direct and automatable methodology for characterising ionic series and the hierarchy of all product ions of an MS spectrum.Two MS/MS with ion mobility experiments have been designed: in the first, the intrinsic mobility of each ion is estimated, and in the second experiment, distributions of the ionic intensity of product ions fragmented after IMS separations are recorded. These mobilograms are aligned using the coshift algorithm and mathematically fitted using Classical Least Squares (CLS) to determine the mobility contributions from their precursor ions.Despite some limitations when studying low intensity ions and ions with similar ion mobility, CLS fitting improves the usage of IMS coupled with accurate mass spectrometry as a complementary tool in the study of MS fragmentation mechanisms and more notably, it offers an automatable and efficient alternative to MS3 experiments.
Keywords: Ion mobility; Flavonoids; Phenolic compounds; Structural characterization; Mass spectrometry; Cross section
Microdialysate analysis of monoamine neurotransmitters—A versatile and sensitive LC–MS/MS method by S. Greco; W. Danysz; A. Zivkovic; R. Gross; H. Stark (pp. 65-72).
Display Omitted► Novel LC/MS/MS method for quantification of neurotransmitter in microdialysate. ► The validated method is based on a pre-column derivatization. ► The method is suitable for high throughput analysis. ► High selectivity and sensitivity. ► Changes in neurotransmitter concentrations were monitored in the striatum of rats.We have developed and validated a sensitive method for the simultaneous determination of some monoamine neurotransmitters like dopamine (DA), norepinephrine (NE) and serotonin (5-HT) in rat brain microdialysate using high-performance liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). Sensitivity enhancement has been achieved by amine derivatization with the reagent (5- N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) under mild conditions. The use of the selected reaction monitoring (SRM) mode has allowed detection of the analytes at a concentration of 30pM (lower limit of quantification, LLOQ, signal-to-noise ratio higher than 5) with an accuracy of ≤3.80% and a precision of ±7.39 (%CV) for all neurotransmitters. Derivatization improves resolution and chromatographic retention times (3min) by lipophilization. Linearity has been good ( R>0.99) over a large concentration range (30–50,000pM). The intra and inter-batch accuracy and precision were not greater than 4.8% and 6.4%, respectively. Therefore, the method was successfully applied for monitoring the concentration changes of neurotransmitters in microdialysis samples deriving from striatum rat brain region after amphetamine administration (3mgkg−1, i.p.).
Keywords: Liquid chromatography–tandem mass spectrometry; Derivatization; High-throughput; Neurotransmitter; Brain microdialysis; Rat; Striatum
Structural studies on archaeal phytanyl-ether lipids isolated from membranes of extreme halophiles by linear ion-trap multiple-stage tandem mass spectrometry with electrospray ionization by Fong-Fu Hsu; Simona Lobasso; John Turk; Angela Corcelli (pp. 73-85).
Archareal bisphosphatidylglycerol (BPG) (I) and sulfo-triglycosyl-diether (S-TGD-1) (II) are unique in that they consist of phytanyl substituents ether linked to the glycerol backbone.Display Omitted► Characterization of archaeal phytanyl ether lipids of extreme halophiles was described. ► Multiple stage (MS n) linear ion-trap high resolution mass spectrometry was used. ► Structurally informative ions defined phytanyl ether chain were observed. ► Unique losses of internal hexose and of glycerol were observed. ► Fragmentation processes leading to the ion formation were proposed.The structures of archaeal glycerophospholipids and glycolipids are unique in that they consist of phytanyl substituents ether linked to the glycerol backbone, imparting stability to the molecules. In this contribution, we described multiple-stage linear ion-trap combined with high resolution mass spectrometry toward structural characterization of this lipid family desorbed as lithiated adduct ions or as the [M−H]− and [M−2H]2− ions by ESI. MS n on various forms of the lithiated adduct ions yielded rich structurally informative ions leading to complete structure identification of this lipid family, including the location of the methyl branches of the phytanyl chain. By contrast, structural information deriving from MS n on the [M−H]− and [M−2H]2− ions is not complete. The fragmentation pathways in an ion-trap, including unusual internal loss of glycerol moiety and internal loss of hexose found for this lipid family were proposed. This mass spectrometric approach provides a simple tool to facilitate confident characterization of this unique lipid family.
Keywords: Abbreviations; ESI-MS; electrospray ionization-MS; HRMS; high resolution mass spectrometry; LIT; linear ion-trapArchaea; Archaeal phospholipids; Archaeal glycolipids; Cardiolipin; Isopranoid chains; Halobacteriaceae
Fast screening of ketamine in biological samples based on molecularly imprinted photonic hydrogels by Liang Meng; Pinjia Meng; Qingqing Zhang; Yanji Wang (pp. 86-94).
A novel label-free colorimetric chemosensor: with the increase in the concentration of ketamine, the Bragg diffraction peak of MIPHs gradually shifted to the longer wavelength region. Accompanying the peak shift, the color change of MIPHs was also observed obviously: from green to red.Display Omitted► We developed the label-free colorimetric MIPHs for handy and fast screening of ketamine. ► The obvious color change of MIPHs was observed upon ketamine. ► The MIPHs exhibited good sensing abilities in an aqueous environment. ► The sensing mechanisms of the water-compatible MIPHs were investigated. ► The MIPHs were employed to screening ketamine in real biological samples.A novel label-free colorimetric chemosensor was developed for handy and fast screening of ketamine with high sensitivity and specificity based on molecularly imprinted photonic hydrogels (MIPHs) that combined the colloidal-crystal with molecular imprinting technique. The unique inverse opal arrays with a thin polymer wall in which the imprinted nanocavities of ketamine moleculars distributed allowed high sensitive, quick responsive, specific detection of the target analyte, and good regenerating ability in an aqueous environment. Due to the hierarchical inverse opal structural characteristics, the specific ketamine molecular recognition process can induce obvious swelling of the MIPHs to be directly transferred into visually perceptible optical signal (change in color) which can be detected by the naked eye through Bragg diffractive shifts of ordered macroporous arrays. In order to enhance the recognition ability in aqueous environments, the MIPHs were designed as water-compatible and synthesized in a water–methanol system. The molecular recognition mechanisms were investigated. The proposed MIPHs were successfully employed to screen trace level ketamine in human urine and saliva samples, exhibiting high sensitivity, rapid response, and specificity in the complex matrix. The smart chemosensor can provide an effective alternative for fast screening of ketamine on the spot for forensic investigations.
Keywords: Molecularly imprinting; Photonic crystal; Ketamine; Chemosensor; Water-phase recognition; Biological samples
A novel Lu3+ fluorescent nano-chemosensor using new functionalized mesoporous structures by Morteza Hosseini; Mohammad Reza Ganjali; Zahra Rafiei-Sarmazdeh; Farnoush Faridbod; Hassan Goldooz; Alireza Badiei; Parviz Nourozi; Ghodsi Mohammadi Ziarani (pp. 95-101).
A novel Lu3+ sensitive fluorescent chemosensor is constructed through the preparation of 8-hydroxyquinoline functionalized mesoporous silica with ordered hexagonal array structure (LUS-SPS-Q). Fluorescence measurements revealed that the emission intensity of the Lu3+-bound mesoporous material increases significantly upon addition of various concentrations of Lu3+, while the mono-, di-, trivalent cations result in either unchanged or weakened intensities.Display Omitted► 8-Hydroxyquinoline functionalized mesoporous silica is introduced as a selective fluorescent probe for lutetium ions. ► Fluorescent intensity of the chemical probe enhances upon binding to lutetium ions. ► Fluorescence measurements were done in a suspension of mesoporous silica in aqueous solution.A new Lu3+ sensitive fluorescent chemosensor is designed using 8-hydroxyquinoline functionalized mesoporous silica with highly ordered structure (LUS-SPS-Q). The characterization of LUS-SPS-Q showed that the organized structure has been preserved after the post grafting procedure. The synthesized material showed a selective interaction with Lu3+ ion, most probably due to the presence of the fluorophore moiety at its surface. The emission intensity of the Lu3+-bound mesoporous material increases with an increase in concentrations of Lu3+ ion. Addition of other mono-, di-, trivalent ions resulted in insignificant change in the fluorescent intensity. The enhancement of fluorescence is attributed to the strong covalent binding of Lu3+ ion. The linear response range of Lu3+ chemo-sensor was from 1.6×10−7 to 1.0×10−5molL−1. The Limit of detection obtained was 8.2×10−8molL−1 and the pH range which the proposed chemo-sensor can be applied was 3.3–8.3.
Keywords: Lutetium; Fluorescent; Mesoporous; Enhancing; Nano-chemosensor
Simultaneous detection of lactate and glucose by integrated printed circuit board based array sensing chip by Xuelian Li; Jianfeng Zang; Yingshuai Liu; Zhisong Lu; Qing Li; Chang Ming Li (pp. 102-107).
Display Omitted► An integrated printed circuit board (PCB) based array sensing chip was developed. ► Simultaneous detection of lactate and glucose in serum has been demonstrated. ► The array electronic biochip has high signal to noise ratio and high sensitivity. ► Additional electrodes were designed on the chip to correct interferences.An integrated printed circuit board (PCB) based array sensing chip was developed to simultaneously detect lactate and glucose in mouse serum. The novelty of the chip relies on a concept demonstration of inexpensive high-throughput electronic biochip, a chip design for high signal to noise ratio and high sensitivity by construction of positively charged chitosan/redox polymer Polyvinylimidazole-Os (PVI-Os)/carbon nanotube (CNT) composite sensing platform, in which the positively charged chitosan/PVI-Os is mediator and electrostatically immobilizes the negatively charged enzyme, while CNTs function as an essential cross-linker to network PVI-Os and chitosan due to its negative charged nature. Additional electrodes on the chip with the same sensing layer but without enzymes were prepared to correct the interferences for high specificity. Low detection limits of 0.6μM and 5μM were achieved for lactate and glucose, respectively. This work could be extended to inexpensive array sensing chips with high sensitivity, good specificity and high reproducibility for various sensor applications.
Keywords: Printed circuit board; Biosensor; Simultaneous detection; Lactate; Glucose; Interference free
Separation and characterisation of beta2-microglobulin folding conformers by ion-exchange liquid chromatography and ion-exchange liquid chromatography–mass spectrometry by Laura Bertoletti; Luca Regazzoni; Giancarlo Aldini; Raffaella Colombo; Franco Abballe; Gabriele Caccialanza; Ersilia De Lorenzi (pp. 108-114).
Display Omitted► We present LC–UV and LC–MS methods to separate protein conformations. ► We demonstrate that protein conformations are at dynamic equilibrium. ► Strong anion exchange chromatography–ESI-MS does not alter β2-microglobulin conformation. ► Charge state distributions characterise the folding state of the separated species. ► The separated species can be molecular targets of drug-like molecules.In this work we present for the first time the use of ion-exchange liquid chromatography to separate the native form and a partially structured intermediate of the folding of the amyloidogenic protein beta2-microglobulin. Using a strong anion-exchange column that accounts for the differences in charge exposure of the two conformers, a LC–UV method is initially optimised in terms of mobile phase pH, composition and temperature. The preferred mobile phase conditions that afford useful information were found to be 35mM ammonium formate, pH 7.4 at 25°C. The dynamic equilibrium of the two species is demonstrated upon increasing the concentration of acetonitrile in the protein sample. Then, the chromatographic method is transferred to MS detection and the respective charge state distributions of the separated conformers are identified. The LC–MS results demonstrate that one of the conformers is partially unfolded, compared with the native and more compact species. The correspondence with previous results obtained in free solution by capillary electrophoresis suggest that strong ion exchange LC–MS does not alter beta2-microglobulin conformation and maintains the dynamic equilibrium already observed between the native protein and its folding intermediate.
Keywords: Abbreviations; β; 2; -m; beta; 2; -microglobulin; CSD; charge state distributionBeta; 2; -microglobulin; Ion-exchange chromatography; Mass spectrometry; Amyloidosis; Protein conformers; Protein folding