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Analytica Chimica Acta (v.722, #)

Editorial Board (pp. iii).

Flower-like self-assembly of gold nanoparticles for highly sensitive electrochemical detection of chromium(VI) by Ruizhuo Ouyang; Stefanie A. Bragg; James Q. Chambers; Zi-Ling Xue (pp. 1-7).

Display Omitted► Fabrication of a flower-like self-assembly of two AuNP layers on a GCE. ► Cr(VI) detection: 10–1200ngL⁻¹ concentration range; 2.9ngL⁻¹ detection limit. ► The 1st AuNP layer on the GCE surface as anchors for a thiol sol–gel film. ► The sol–gel film link the 1st AuNP layer to the 2nd AuNP layer. ► Functionalization of the 2nd AuNP layer by a thiol pyridinium for HCrO₄⁻ detection.We report here the fabrication of a flower-like self-assembly of gold nanoparticles (AuNPs) on a glassy carbon electrode (GCE) as a highly sensitive platform for ultratrace Cr(VI) detection. Two AuNP layers are used in the current approach, in which the first is electroplated on the GCE surface as anchors for binding to an overcoated thiol sol–gel film derived from 3-mercaptopropyltrimethoxysilane (MPTS). The second AuNP layer is then self-assembled on the surface of the sol–gel film, forming flower-like gold nanoelectrodes enlarging the electrode surface. When functionalized by a thiol pyridinium, the fabricated electrode displays a well-defined peak for selective Cr(VI) reduction with an unusually large, linear concentration range of 10–1200ngL⁻¹ and a low detection limit of 2.9ngL⁻¹. In comparison to previous approaches using MPTS and AuNPs on Au electrodes, the current work expands the use of AuNPs to the GCE. Subsequent functionalization of the secondary AuNPs by a thiol pyridinium and adsorption/preconcentration of Cr(VI) lead to the unusually large detection range and high sensitivity. The stepwise preparation of the electrode has been characterized by electrochemical impedance spectroscopy (EIS), scanning electronic microscopy (SEM), and IR. The newly designed electrode exhibits good stability, and has been successfully employed to measure chromium in a pre-treated blood sample. The method demonstrates acceptable fabrication reproducibility and accuracy.

Keywords: Gold nanoparticles; Self-assembly; Glassy carbon electrode; Sol–gel; Thiol pyridinium; Chromium detection

Analytical procedures for the determination of emerging organic contaminants in plant material: A review by Víctor Matamoros; Diana Calderón-Preciado; Carmen Domínguez; Josep M. Bayona (pp. 8-20).

Display Omitted► This work reviews all available analytical methodologies for the determination of emerging contaminants in plant tissues. ► A good sampling strategy is necessary to obtain reliable results. ► Extraction and clean-up are widely used in sample pre-treatment. ► LC and GC coupled to MS are normally necessary. ► New developments and configurations are presented.In this review, recent developments for the determination of emerging organic contaminants (EOCs) in plant tissues are discussed focusing on the homogenization, extraction and determination steps involved. Eleven classes of EOCs, namely antibiotics, analgesics, antiepileptics, antidepressants, antiseptics, plasticizers, fragrances, surfactants, flame retardants, and phenoxy acid herbicides, have been evaluated. Methods are critically reviewed in terms of all the analytical steps involved in the analysis, sampling and sample preparation, separation, and the detection strategies employed. The extraction from tissue samples was performed in most cases by solid–liquid extraction, whereas the clean-up was performed by solid-phase extraction. The identification and quantification of EOCs in crops from the agricultural field (i.e. parts per billion range) is usually performed by using mass spectrometry techniques such as single quadrupole mass spectrometry or tandem mass spectrometry coupled to high resolution chromatographic techniques. Enzyme-linked immunosorbent assays are more rarely used. New developments such as in vivo solid-phase microextraction (SPME) and the assessment of the bioavailability–bioaccesibility of contaminants in crops are shown. The main scope of this review is to critically evaluate the current state of the art of the analytical techniques used and to identify the research needs in the determination of EOCs in crops.

Keywords: Emerging organic contaminants; Sample preparation; Plant uptake; In vivo SPME; Mass spectrometry; Extraction

Submicron hard X-ray fluorescence imaging of synthetic elements by Mark P. Jensen; Baikuntha P. Aryal; Drew Gorman-Lewis; Tatjana Paunesku; Barry Lai; Stefan Vogt; Gayle E. Woloschak (pp. 21-28).

Display Omitted► Actinide elements are mapped with L-edge X-rays and better than 400nm resolution. ► A typical detection limit was 2.9×10⁻²⁰molesPuμm⁻². ► XANES measurements provide chemical information in 0.1μm² spots. ► Selection of materials for encapsulation is important for avoiding interferences.Synchrotron-based X-ray fluorescence microscopy (XFM) using hard X-rays focused into sub-micron spots is a powerful technique for elemental quantification and mapping, as well as microspectroscopic measurements such as μ-XANES (X-ray absorption near edge structure). We have used XFM to image and simultaneously quantify the transuranic element plutonium at the L₃ or L₂-edge as well as Th and lighter biologically essential elements in individual rat pheochromocytoma (PC12) cells after exposure to the long-lived plutonium isotope²⁴²Pu. Elemental maps demonstrate that plutonium localizes principally in the cytoplasm of the cells and avoids the cell nucleus, which is marked by the highest concentrations of phosphorus and zinc, under the conditions of our experiments. The minimum detection limit under typical acquisition conditions with an incident X-ray energy of 18keV for an average 202μm² cell is 1.4fg Pu or 2.9×10⁻²⁰molesPuμm⁻², which is similar to the detection limit of K-edge XFM of transition metals at 10keV. Copper electron microscopy grids were used to avoid interference from gold X-ray emissions, but traces of strontium present in naturally occurring calcium can still interfere with plutonium detection using its L_α X-ray emission.

Keywords: X-ray fluorescence microscopy; Chemical imaging; Actinide; Plutonium; X-ray absorption near edge structure

Determination of copper, iron, nickel and zinc in ethanol fuel by energy dispersive X-ray fluorescence after pre-concentration on chromatography paper by Leonardo Sena Gomes Teixeira; Elenir Souza Santos; Luana Sena Nunes (pp. 29-33).

Display Omitted► A procedure is proposed for analyzing metal ions in ethanol fuel samples by EDXRF after a preconcentration step. ► The preconcentration process involves analyte retention on chromatography paper. ► The method does not require the sample to be subjected to time-consuming pretreatment.This paper presents an alternative analytical method employing energy dispersive X-ray fluorescence (EDXRF) to determine copper, iron, nickel and zinc ions in ethanol fuel samples after a pre-concentration procedure. Our pre-concentration strategy utilizes analyte retention on cation exchange chromatography paper, a convenient substrate for direct EDXRF measurements. The repeatability, expressed in terms of RSD of standard solutions containing 0.25μgmL⁻¹ of Cu, Fe, Ni and Zn, and calculated from fifteen consecutive measurements, was 2.5, 2.8, 3.0, and 2.7%, respectively. The limits of detection (LOD), defined as the analyte concentration that gives a response equivalent to three times the standard deviation of the blank ( n=10), were found to be 13, 15, 15 and 12μgL⁻¹ for Cu, Fe, Ni and Zn, respectively. The proposed method was applied to Cu, Fe, Ni and Zn determination in hydrated ethanol fuel samples collected from different gas stations.

Keywords: Ethanol fuel; EDXRF; Metals determination; Chromatography paper

Combination of chromatographic and chemometric methods to study the interactions between DNA strands by Sara Ruiz-Castelar; Antonio Checa; Raimundo Gargallo; Joaquim Jaumot (pp. 34-42).

Display Omitted► Coupled chromatography and chemometrics to study complex DNA structures formation. ► Size-exclusion chromatography is used to separate different DNA structures. ► MCR-ALS applied to study the three-way data sets obtained by SEC-PDA. ► Study of the equilibria between duplex and quadruplex DNA structures.This work describes the combination of size-exclusion chromatography and chemometric resolution methods to study the formation of complex DNA structures from individual strands. This combined procedure has been applied to two different experimental data. Firstly, the formation of an intermolecular Watson–Crick duplex structure formed by the individual unstructured strands. Secondly, the competition between the intermolecular Watson–Crick duplex and intramolecular quadruplex structures formed by two sequences found in the hTERT gene has been studied.The analysis of the recorded chromatograms at just one single wavelength may not always be enough to confirm the existence of a higher order structure. In these cases, recording the entire spectrum at each point of the chromatogram and applying appropriate chemometric resolution methods has shown to be a useful tool to resolve and quantify the contribution of all DNA structures to the analytical signal. In this work, the Multivariate Curve Resolution based on Alternating Least Squares (MCR-ALS) has been used to analyze the three-way data sets acquired along chromatographic runs.

Keywords: Abbreviations; hTERT; human Telomerase Reverse Transcriptase; MCR-ALS; Multivariate Curve Resolution Alternating Least Squares; CD; circular dichroism; HPLC-PDA; High-Performance Liquid Chromatography Photo Diode Array Detector; SEC; Size Exclusion ChromatographySize exclusion chromatography; Chemometrics; Nucleic acids; Quadruplex; Multivariate Curve Resolution

Assessment of a geochemical extraction procedure to determine the solid phase fractionation and bioaccessibility of potentially harmful elements in soils: A case study using the NIST 2710 reference soil by Joanna Wragg; Mark Cave (pp. 43-54).

Display Omitted► Three mineral acid extractions regimes were used. ► Self-modelling mixture resolution identified 12 distinct components. ► The human ingestion bioaccessible As, Cd and Pb fractions were measured. ► The bioaccessible fractions were assigned to specific physico-chemical components. ► The extraction scheme using a mixture of HCl and HNO₃ was found to work best.Three mineral acid sequential extraction regimes (HNO₃ only, HNO₃ followed by HCl and aqua regia) were applied to the NIST 2710 contaminated reference soil. The major and trace element chemical analysis data from the extractions were subjected to a chemometric self-modelling mixture resolution procedure which identified that 12 distinct physico-chemical components were extracted. The fractionation of As, Cd, Ni and Pb between these components were determined. Tentative assignments of the mineralogical sources of the components were made. The human ingestion bioaccessible fraction of As, Cd and Pb were determined using the in vitro BARGE UBM bioaccessibility test and were found to be 51.6%, 68.0% and 68.4% respectively. The relationship between the lability of the physico-chemical components and the bioaccessible fraction of the soils was investigated and the bioaccessible fractions were assigned to specific components. The extraction scheme using aqua regia was found to be the most suitable as it was the only one which extracted the iron sulphide phase in the soil.

Keywords: Bioaccessibility; Sequential extraction; UK risk assessment; PHE; Arsenic; Cadmium; Lead; Nickel

Electro membrane extraction of sodium diclofenac as an acidic compound from wastewater, urine, bovine milk, and plasma samples and quantification by high-performance liquid chromatography by Saied Saeed Hosseiny Davarani; Ahmad Pourahadi; Saeed Nojavan; Mohammad Hossein Banitaba; Mahnaz Nasiri-Aghdam (pp. 55-62).

Display Omitted► EME was used for determination of an acidic compound in biological samples. ► Extraction was done in low voltage (20V) and it was fast (5min). ► Good recovery (95%) and good enrichment factor (67) were obtained. ► Presence of salt in plasma caused low recovery in comparison with pure water. ► Optimized method was used for determination of diclofenac in different samples.Electro membrane extraction (EME) as a new microextraction method was applied for extraction of sodium diclofenac (SDF) as an acidic compound from wastewater, urine, bovine milk and plasma samples. Under applied potential of 20V during the extraction, SDF migrated from a 2.1mL of sample solution (1mM NaOH), through a supported liquid membrane (SLM), into a 30μL acceptor solution (10mM NaOH), exist inside the lumen of the hollow fiber. The negative electrode was placed in the donor solution, and the positive electrode was placed in the acceptor solution. 1-octanol was immobilized in the pores of a porous hollow fiber of polypropylene as SLM. Then the extract was analyzed by means of high-performance liquid chromatography (HPLC) with UV-detection for quantification of SDF. Best results were obtained using a phosphate running electrolyte (10mM, pH 2.5). The ranges of quantitation for different samples were 8–500ngmL⁻¹. Intra- and inter-day RSDs were less than 14.5%. Under the optimized conditions, the preconcentration factors were between 31 and 66 and also the limit of detections (LODs) ranged from 2.7ngmL⁻¹ to 5ngmL⁻¹ in different samples. This procedure was applied to determine SDF in wastewater, bovine milk, urine and plasma samples (spiked and real samples). Extraction recoveries for different samples were between 44–95% after 5min of extraction.

Keywords: Abbreviations; EME; electro membrane extraction; SLM; supported liquid membrane; SDF; sodium diclofenac; NPOE; 2-nitrophenyl octyletherElectro membrane extraction; Liquid chromatography; Wastewater; Bovine milk; Urine; Plasma; Sodium diclofenac

Method development for proteome stabilization in human saliva by Hua Xiao; David T.W. Wong (pp. 63-69).

Display Omitted► We developed a simple method for the stabilization of salivary proteome at RT. ► Stored saliva samples could be used for different applications. ► This optimized method is a milestone for future salivary diagnostics.Human saliva is a biological fluid with emerging early detection and diagnostic potentials. However, the salivary proteome suffers from rapid degradation and thus compromises its translational and clinical utilities. Therefore, easy, reliable and practical methods are urgently required for the storage of human saliva samples. In this study, saliva samples from healthy subjects were collected and stored at room temperature (RT) and 4°C for different lengths of time with and without specific protein stabilization treatments. SDS-PAGE was run to compare the protein profiling between samples. Reference proteins, β-actin and interleukin-1 β (IL1β), were chosen to evaluate salivary protein stability. Immunoassay was used for the detection of these target proteins. All data was compared with the positive control that had been kept at −80°C. The results show that the salivary proteome that has been stored at 4°C with added protease inhibitors was stable for approximately two weeks without significant degradation. By adding ethanol to the samples, the salivary proteome was stabilized at RT. After optimization, a simple, robust and convenient method is developed for the stabilization of proteins in human saliva that does not affect the downstream translational and clinical applications. The salivary proteome could be stabilized without significant degradation by adding ethanol at RT for about two weeks. This optimized method could greatly accelerate the clinical usage of saliva for future diagnosis.

Keywords: Human saliva; Proteome; Protein stabilization; Immunoassay; Clinical application

Novel methods for the quantification of (2 E)-hexadecenal by liquid chromatography with detection by either ESI QTOF tandem mass spectrometry or fluorescence measurement by Anja Lüth; Corinna Neuber; Burkhard Kleuser (pp. 70-79).

Display Omitted► Very sensitive tandem-MS method for (2 E)-hexadecenal. ► Easily practicable HPLC/fluorescence detection method for (2 E)-hexadecenal. ► HPLC/fluorescence: for the first time in the present sphingolipid research. ► Derivatization with DAIH to generate a comfortable analyzable azine. ► Determination of (2 E)-hexadecenal level in biological samples for the first time ever.Sphingosine-1-phosphate lyase (SPL) is the only known enzyme that irreversibly cleaves sphingosine-1-phosphate (S1P) into phosphoethanolamine and (2 E)-hexadecenal during the final step of sphingolipid catabolism. Because S1P is involved in a wide range of physiological and diseased processes, determining the activity of the degrading enzyme is of great interest. Therefore, we developed two procedures based on liquid chromatography (LC) for analysing (2 E)-hexadecenal, which is one of the two S1P degradation products. After separation, two different quantification methods were performed, tandem mass spectrometry (MS) and fluorescence detection. However, (2 E)-hexadecenal as a long-chain aldehyde is not ionisable by electrospray ionisation (ESI) for MS quantification and has an insufficient number of corresponding double bonds for fluorescence detection. Therefore, we investigated 2-diphenylacetyl-1,3-indandione-1-hydrazone (DAIH) as a derivatisation reagent. DAIH transforms the aldehyde into an ionisable and fluorescent analogue for quantitative analysis. Our conditions were optimised to obtain the outstanding limit of detection (LOD) of 1fmol per sample (30μL) for LC–MS/MS and 0.75pmol per sample (200μL) for LC determination with fluorescence detection. We developed an extraction procedure to separate and concentrate (2 E)-hexadecenal from biological samples for these measurements. To confirm our new methods, we analysed the (2 E)-hexadecenal level of different cell lines and human plasma for the first time ever. Furthermore, we treated HT-29 cells with different concentrations of 4-deoxypyridoxine (DOP), which competitively inhibits pyridoxal-5-phosphate (P5P), an essential cofactor for SPL activity, and observed a significant decrease in (2 E)-hexadecenal relative to the untreated cells.

Keywords: (2; E; )-Hexadecenal; Sphingosine-1-phosphate lyase; Derivatisation; Tandem mass spectrometry; Fluorescence

Determination of triacylglycerol regioisomers using electrospray ionization-quadrupole ion trap mass spectrometry with a kinetic method by Nathalie L. Leveque; Akwasi Acheampong; Sylvie Heron; Alain Tchapla (pp. 80-86).

Display Omitted► This manuscript presents the methodology to distinguish the triacylglycerols regiospecificity. ► The kinetic method associated with the standard addition method was applied. ► The quantification of TAG isomers is possible even if only one TAG standard is available.The kinetic method was applied to differentiate and quantify mixtures of regioisomeric triacylglycerols (TAGs) by generating and mass selecting alkali ion bound metal dimeric clusters with a TAG chosen as reference (ref) and examining their competitive dissociations in a quadrupole ion trap mass spectrometer. This methodology readily distinguished pairs of regioisomers (AAB/ABA) such as LLO/LOL, OOP/OPO and SSP/SPS and consequently distinguished sn-1/ sn-3, sn-2 substituents on the glycerol backbone. The dimeric complex ions [ref, Li, TAG_{(AAB and/or ABA)}]⁺ generated by electrospray ionization mass spectrometry were subjected to collision induced dissociation causing competitive loss of either the neutral TAG reference (ref) leading to [Li(AAB and/or ABA)]⁺ or the neutral TAG molecule (TAG_{(AAB and/or ABA)}) leading to [ref, Li]⁺. The ratio of the two competitive dissociation rates, defined by the product ion branching ratio ( R_iso), was related via the kinetic method to the regioisomeric composition of the investigated TAG mixture. In this work, a linear correlation was established between composition of the mixture of each TAG regioisomer and the logarithm of the branching ratio for competitive fragmentation. Depending on the availability of at least one TAG regioisomer as standard, the kinetic method and the standard additions method led to the quantitative analysis of natural TAG mixtures.

Keywords: Kinetic method; Mass spectrometry; Regiospecificity; Triacylglycerols

Nuclear magnetic resonance-based metabolomics for prediction of gastric damage induced by indomethacin in rats by So Young Um; Jung Hyun Park; Myeon Woo Chung; Kyu-Bong Kim; Seon Hwa Kim; Ki Hwan Choi; Hwa Jeong Lee (pp. 87-94).

.Display Omitted► NMR based metabolomics – gastric damage by indomethacin. ► Pattern recognition analysis was performed to biomarkers of gastric damage. ► 2-Oxoglutarate, acetate, taurine and hippurate were selected as putative biomarkers. ► The gastric damage induced by NSAIDs can be screened in the preclinical step of drug.Non-steroidal anti-inflammatory drugs (NSAIDs) have side effects including gastric erosions, ulceration and bleeding. In this study, pattern recognition analysis of the¹H-nuclear magnetic resonance (NMR) spectra of urine was performed to develop surrogate biomarkers related to the gastrointestinal (GI) damage induced by indomethacin in rats. Urine was collected for 5h after oral administration of indomethacin (25mgkg⁻¹) or co-administration with cimetidine (100mgkg⁻¹), which protects against GI damage. The¹H-NMR urine spectra were divided into spectral bins (0.04ppm) for global profiling, and 36 endogenous metabolites were assigned for targeted profiling. The level of gastric damage in each animal was also determined. Indomethacin caused severe gastric damage; however, indomethacin administered with cimetidine did not. Simultaneously, the patterns of changes in their endogenous metabolites were different. Multivariate data analyses were carried out to recognize the spectral pattern of endogenous metabolites related to indomethacin using partial least square-discrimination analysis. In targeted profiling, a few endogenous metabolites, 2-oxoglutarate, acetate, taurine and hippurate, were selected as putative biomarkers for the gastric damage induced by indomethacin. These metabolites changed depending on the degree of GI damage, although the same dose of indomethacin (10mgkg⁻¹) was administered to rats. The results of global and targeted profiling suggest that the gastric damage induced by NSAIDs can be screened in the preclinical stage of drug development using a NMR based metabolomics approach.

Keywords: Metabolomics; Indomethacin; Cimetidine· Gastric damage; NMR; Biomarker

Application of europium(III) chelates-bonded silica nanoparticle in time-resolved immunofluorometric detection assay for human thyroid stimulating hormone by Yulin Zhou; Xiaohu Xia; Ye Xu; Wei Ke; Wei Yang; Qingge Li (pp. 95-99).

Display Omitted► A rapid and ultrasensitive TSH immunoassay was developed using fluorescent silica nanoparticles-based TrIFA. ► The assay is of high sensitivity with short period time request. ► method can be potentially used at hospitals for daily clinical practice in hTSH screening.Eu(III) chelate-bonded silica nanoparticle was used as a fluorescent label to develop a highly sensitive time-resolved immunofluorometric assay (TrIFA) for human thyroid stimulating hormone (hTSH). The limit of detection of the assay calculated according to the 2SD method was 0.0007mIUL⁻¹ and became 0.003mIUL⁻¹ when serum-based matrix was used for calibrators, indicating that this TrIFA is comparable with the most sensitive assays. The linear range was from 0.005 to 100mIUL⁻¹ of hTSH with coefficient of variation between 1.9% and 8.3%. The correlation study using 204 blood spot samples from newborns showed that the results from this new method were coincident with that of the commercial dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) system, with a correlation coefficient of 0.938. The fluorescent nanoparticle label allows directly reading the fluorescent signal, omitting the signal development step required for the DELFIA system, and the whole procedure of this assay is fulfilled within 2h. Thus, we developed a novel, sensitive, quantitative and simple nanoparticle label-based TrIFA assay, suitable for routine application in hTSH screening of neonatal hypothyroidism.

Keywords: Europium(III) chelate; Silica nanoparticle; Human thyroid stimulating hormone; Time-resolved immunofluorometric assay

Electrochemical immunoassay of benzo[a]pyrene based on dual amplification strategy of electron-accelerated Fe₃O₄/polyaniline platform and multi-enzyme-functionalized carbon sphere label by Mouhong Lin; Yingju Liu; Zihong Sun; Shenglai Zhang; Zhuohong Yang; Chunlin Ni (pp. 100-106).

Schematic representation of Fe₃O₄/PANI/Nafion-based immunosensor using multi-HRP-HCS-Ab₂ bioconjugates as labels.Display Omitted► An electrochemical immunosensor for high sensitive detection of BaP. ► A dual amplification strategy by Fe₃O₄/PANI/Nafion film and multi-HRP-HCS-Ab₂ label. ► An accelerated electron transfer pathway by the Fe₃O₄/PANI/Nafion film.An electrochemical immunosensor, basing on a dual amplification strategy by employing a biocompatible Fe₃O₄/polyaniline/Nafion (Fe₃O₄/PANI/Nafion) layer as sensor platform and multi-enzyme-antibody functionalized highly-carbonized spheres (multi-HRP-HCS-Ab₂) as label, was constructed for sensitive detection of benzo[a]pyrene (BaP). The stable film, Fe₃O₄/PANI/Nafion, can not only immobilize biomolecules, but also catalyze the reduction of hydrogen peroxide, indicating an accelerated electron transfer pathway of the platform. The experimental conditions, including the concentration of Nafion, concentration of Fe₃O₄/polyaniline (Fe₃O₄/PANI), pH of the detection solution and concentrations of biomolecules, were studied in detail. Basing on a competitive immunoassay, the current change was proportional to the logarithm of BaP concentration in the range of 8pM and 2nM with the detection limit of 4pM. The proposed immunosensor exhibited acceptable reproducibility and stability. This new type of dual amplification strategy may provide potential applications for the detection of environmental pollutants.

Keywords: Immunosensor; Benzo[a]pyrene; Fe; ₃; O; ₄; /polyaniline; Carbon nanospheres

Dipstick based immunochemiluminescence biosensor for the analysis of vitamin B₁₂ in energy drinks: A novel approach by L.S. Selvakumar; M.S. Thakur (pp. 107-113).

(a) Schematic representation of immunochemiluminescence based dipstick technique for detection of vitamin B₁₂. (b) Enzymatic dephosphorylation of dioxetane by alkaline phosphatase.Display Omitted► Dipstick based immunochemiluminescence biosensor proposed for vitamin B₁₂ analysis. ► The limit of detection of vitamin B₁₂ is 1ngmL⁻¹ and applied in energy drinks. ► Chemiluminescence generated was inversely proportional to vitamin B₁₂ concentration. ► Chemiluminescence analytical procedure was compared with ELISA. ► Alkaline phosphatase was stable chemiluminescent enzyme than Horse Radish Peroxidase.In this article, we describe a dipstick based immunochemiluminescence (immuno-CL) biosensor for the detection of vitamin B₁₂ in energy drinks. The method is a direct competitive type format involving the immobilization of vitamin B₁₂ antibody on nitrocellulose membrane (NC) followed by treatment with vitamin B₁₂ and vitamin B₁₂–alkaline phosphatase conjugate to facilitate the competitive binding. The dipstick was further treated with substrate disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenyl phosphate (CDP-Star) to generate chemiluminescence (CL). The number of photons generated was inversely proportional to the vitamin B₁₂ concentration. After systematic optimization, the limit of detection was 1ngmL⁻¹. The coefficient of variation was below 0.2% for both intra- and inter-assay precision. Vitamin B₁₂ was extracted from energy drinks with recovery ranged from 90 to 99.4%. Two different energy drinks samples were analyzed, and a good correlation was observed when the data were compared with a reference enzyme linked immuno sorbent assay (ELISA) method. The developed method is suitable for an accurate, sensitive, and high-throughput screening of vitamin B₁₂ in energy drinks samples. The dipstick technique based on immuno-CL is suitable for the detection of several analyte in food and environmental samples.

Keywords: Biosensor; Dipstick; Immunochemiluminescence; Enzyme linked immunosorbent assay; Vitamin B; ₁₂

Chemiluminescence enzyme immunoassay using magnetic nanoparticles for detection of neuron specific enolase in human serum by Xiaoling Fu; Meng Meng; Yu Zhang; Yongmei Yin; Xiangsheng Zhang; Rimo Xi (pp. 114-118).

Display Omitted► The FITC labeled NSE capture antibody and ALP labeled NSE detection antibody were prepared to develop a sandwich detection format. ► The immune complex formed in aqueous solution and then bound with anti-FITC immobilized magnetic beads for detection of NSE by chemiluminescence intensity. ► The presented method showed high sensitivity and satisfactory recovery and coefficient of variation. ► A linear relationship was obtained for detection results of 120 patients’ sera by the proposed method and traditional chemiluminescence immunoassay.To detect a biomarker for small cell lung carcinoma, neuron specific enolase (NSE), a sensitive and specific chemiluminescence enzyme immunoassay was developed. Fluorescein isothiocyanate (FITC) labeled NSE capture antibody connected with NSE and alkaline phosphatase (ALP) labeled NSE detection antibody in a sandwich-type detection manner. This immune complex was further reacted with anti-FITC coated magnetic beads. In a magnetic field, the complex was enriched, and the sensitivity was thus enhanced. The limit of detection (LOD) of this method was <0.2ngmL⁻¹. The proposed immunoassay was highly selective, and not interfered by hook effect. The recovery was >83.0% and the coefficient of variation was <10.0%. Human sera from 120 patients were tested with the presented and traditional chemiluminescence enzyme immunoassay. An excellent linear relationship was obtained between two techniques. Overall, this immunoassay offers a promising alternative for NSE detection than traditional clinical examinations.

Keywords: Magnetic particle separation; Chemiluminescence enzyme immunoassay; Neuron specific enolase (NSE); Small cell lung carcinoma (SCLC); Neuroblastoma

Nanosensors lost in space. A random walk study of single molecule detection with single-nanopore sensors by Lajos Höfler; Róbert E. Gyurcsányi (pp. 119-126).

Display Omitted► Addressing the problem of single molecule detection with single nanopores. ► Random walk simulations are proposed to assess the “detection limit” of nanosensors. ► Random walk simulations are adapted to account for electrophoretic driving forces. ► Equations are provided for the encounter time in mass transport limited conditions.Nanopores by providing single molecule detection and manipulation are lately in the forefront of life science and nanotechnology research. While single nanopore sensors can detect the residence of even one molecule or nanoparticle within the nanopore, the analytical significance of this process is often misunderstood. A fundamental problem of nanosensors is that their sensing zone is generally infinitesimal with respect of the probed sample volume. Consequently, the probability to have in extremely diluted solutions target molecules or nanoparticles encountering the nanosensor is low. Thus, eventhough the sensor by itself has single molecule detection capability the average time frame in which this occurs is by far not irrelevant for the analysis. In this paper we report on random walk simulations to determine the average time (encounter time) needed by a single molecule to encounter a single nanopore sensor. By assigning the simulation environment with real space and time values a semi-empirical equation for expressing the average encounter time in purely diffusive systems is provided. We also show that random walk simulations can be adapted to evaluate the encounter time in the presence of an external force field acting on the target molecule. As practically relevant application the case of electrophoretically driving DNA strands towards the nanopore sensor is presented and a semi-empirical equation for the encounter time is provided.

Keywords: Electrochemical sensing; Nanopore sensors; Single molecule detection; Random walk; Encounter time; Detection limit

New development in in-capillary electrophoresis techniques for kinetic and inhibition study of enzymes by Hala Nehme; Reine Nehme; Pierre Lafite; Sylvain Routier; Philippe Morin (pp. 127-135).

Display Omitted► We optimized and compared three CE techniques for enzymatic assays. ► We used short-end injection to speed-up electrophoretic analysis (<0.8min). ► We determined kinetic parameters ( V_max, K_m) for β-galactosidase/PNPG. ► We determined inhibition parameters of β-Gal without mixing substrate and inhibitor. ► EMMA is the best technique for enzyme analysis by CE as it is time and sample saving.Enzymes are often quantified by measuring their biological activity. Capillary electrophoresis is gaining its position in this field due to the ongoing trend to miniaturize biochemical assays.The aim of this work was to compare pre-capillary (off-line) and in-capillary electrophoresis techniques for studying enzymatic activity. The β-galactosidase (β-Gal) was chosen as a model enzyme. Each technique was optimized independently in order to decrease analyte consumption (to few tens of nanoliters), incubation time (to few seconds) and analysis time (below 1min). Several experimental parameters (ionic strength of the background electrolyte (BGE) and of the incubation buffer, incubation time, injected volumes, …) were optimized by following peak efficiencies, resolution and repeatability. To monitor the performance of each technique, the catalytic constants ( V_max and K_m) of 4-nitro-phenyl-d-galactopyranoside (PNPG) hydrolysis by β-Gal as well as the inhibition constants ( K_i and IC₅₀) by a competitive inhibitor 2-nitrophenyl-1-thio-β-d-thiogalactopyranoside (ONPTG) were determined. The results obtained were cross compared and were also evaluated by comparison to a standard spectrophotometric method.EMMA proved to be the best technique in terms of sample consumption and speed. The short-end injection was successfully used which speeded-up electrophoretic analysis (<0.8min). It is a very powerful tool for studying enzymatic inhibition. Usually, the inhibitor is injected in the capillary mixed to the substrate especially when both have similar mobilities. We show in this work, for the first time, that combining at-inlet reaction with EMMA-CE allows enzyme inhibition to be realized without any prior mixing of the substrate and the inhibitor. This approach is very interesting for screening inhibitors, rapidly and without excessive substrate consumption.

Keywords: Abbreviations; BGE; background electrolyte; β-Gal; β-galactosidase; EMMA; electrophoretically mediated microanalysis; PMMA; pressure mediated microanalysis; PNPG; 4-nitro-phenyl-; d; -galactopyranoside; PNP; 4-nitrophenol; ONPTG; 2-nitrophenyl-1-thio-β-; d; -thiogalactopyranosideElectrophoretically mediated microanalysis; Pressure mediated microanalysis; Catalytic constants; Inhibition; Screening; Miniaturization

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Analytica Chimica Acta (v.722, #)