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Analytica Chimica Acta (v.724, #)

Editorial Board (pp. iii).

Combining chromatography and chemometrics for the characterization and authentication of fats and oils from triacylglycerol compositional data—A review by Juan M. Bosque-Sendra; Luis Cuadros-Rodríguez; Cristina Ruiz-Samblás; A. Paulina de la Mata (pp. 1-11).

Display Omitted► Relationships between chemometric and chromatography. ► Use of triglyceride profiles and triglyceride chromatographic fingerprints for the characterization of fat and oils. ► Chemometric tools applied to authenticate fat and oils.The characterization and authentication of fats and oils is a subject of great importance for market and health aspects. Identification and quantification of triacylglycerols in fats and oils can be excellent tools for detecting changes in their composition due to the mixtures of these products. Most of the triacylglycerol species present in either fats or oils could be analyzed and identified by chromatographic methods. However, the natural variability of these samples and the possible presence of adulterants require the application of chemometric pattern recognition methods to facilitate the interpretation of the obtained data. In view of the growing interest in this topic, this paper reviews the literature of the application of exploratory and unsupervised/supervised chemometric methods on chromatographic data, using triacylglycerol composition for the characterization and authentication of several foodstuffs such as olive oil, vegetable oils, animal fats, fish oils, milk and dairy products, cocoa and coffee.

Keywords: Abbreviations; ANN; artificial neural network; ANOVA; analysis of variance; APCI; atmospheric-pressure chemical ionization; CAD; charged aerosol detector; CART; classification and regression tree; CB; cocoa butter; CBE; cocoa butter equivalent; CDA; canonical discriminant analysis; CN; carbon number; DA; discriminant analysis; DAG; diacylglycerols; ECN; equivalent carbon number; EDA; exploratory data analysis; EI; electron impact ionization; ELSD; evaporative light scattering detector; ESI; electrospray ionization; FA; fatty acid; FAME; fatty acid methyl ester; FD; fluorescence detector; FID; flame ionization detector; GC; gas chromatography; HCA; hierarchical cluster analysis; HPLC; high performance liquid chromatography; HRGC; high resolution gas chromatography; HTGC; high temperature gas chromatography; iPLS; interval partial least squares; IT; ionic trap; KNN; K-nearest neighbor classification; LDA; linear discriminant analysis; LLL; trilinolein; LLLn; 1,2-linolein-3-linolenin; LLP; 1,2-linolein-3-palmitin; LnPP; 1-linolenin-2-palmitin-3-palmitin; LOO; 1-linolein-2,3-olein; LOP; 1-linolein-2-olein-3-palmitin; MALDI; matrix-assisted laser desorption/ionization; MANOVA; multivariate analysis of variance; MCR; multivariate curve resolution; MLR; multiple linear regressions; MS; mass spectrometry; NP; normal phase; OLA; 1-olein-2-linolein-3-arachidin; OLL; 1-olein-2,3-linolein; OLO; 1,3-olein-2-linolein; OOL; 1,2-olein-3-linolein; OOO; triolein; PCA; principal component analysis; PLL; 1-palmitin-2,3-linolein; PLO; 1-palmitin-2-linolein-3-olein; PLS; partial least squares; PLS-DA; partial least squares-discriminant analysis; POO; 1-palmitin-2,3-olein; PoOO; 1-palmitolein-2,3-olein; RID; refractive index detector; RMSEC; root mean square error of calibration; RMSECV; root mean square error of cross-validation; RMSEP; root mean square error of prediction; RP; reverse phase; SDA; stepwise discriminant analysis; SFC; supercritical fluid chromatography; SFDA; stepwise factorial discriminant analysis; SIMCA; soft independent modeling class analogy; SLS; 1,3-stearin-2-linolein; SOL; 1-stearin-2-olein-3-linolein; SOO; 1-stearin-2,3-olein; SPL; 1-stearin-2-palmitin-3-linolein; TAG; triacylglycerol; TCD; thermal conductivity detector; TIC; total ion current; TLC; thin-layer chromatography; TMS; total mass spectralAuthentication; Triacylglycerols; High temperature gas chromatography; Liquid chromatography; Multivariate data analysis

Locally linear embedding method for dimensionality reduction of tissue sections of endometrial carcinoma by near infrared spectroscopy by Na Qi; Zhuoyong Zhang; Yuhong Xiang; Peter de B. Harrington (pp. 12-19).

Display Omitted► Locally linear embedding (LLE) is introduced first time to the field of spectroscopy as a dimensionality reduction method for feature extraction of near infrared spectra from reflectance measurements of endometrial tissue sections. ► LLE was evaluated and compared with principal component compression (PCC) by using support vector machine (SVM) classifiers. ► The projected difference resolution (PDR) method was used to evaluate the LLE method. ► Some exemplary synthetic data sets were created and NIR spectral data of real tissue samples were collected to verify LLE coupled to SVM for classification. ► LLE combined with the SVM gave better predictions and can effectively extract more discriminating features compared to PCC without scaling.Locally linear embedding (LLE) is introduced here as a nonlinear compression method for near infrared reflectance spectra of endometrial tissue sections. The LLE has been evaluated by using support vector machine (SVM) classifiers and the projected difference resolution (PDR) method. Synthetic data sets devised to resemble near-infrared spectra of tissue samples were used to characterize the performance of the LLE. The LLE was compared using principal component compression (PCC) method to evaluate nonlinear and linear compression. For a set of real tissue samples, if the compressed data were not range-scaled prior to SVM classification, the principal component compressed data gave an average prediction rate of 39±2% while the LLE 94±2%; if range-scaled after compression, the LLE and PCC performed evenly, with maximum average prediction values of 94±2% and 93±2%, respectively. The SVM without compression yielded a classification rate of 92±2%. The prediction accuracy was consistent with PDR results. Without the second derivative preprocessing, the classification rates were 90±3%, 89±2%, and 78±2% for the LLE compressed, the PCC, and no compression classifications by the SVM, respectively.

Keywords: Near infrared spectroscopy; Locally linear embedding; Principal component compression; Support vector machine; Cancer diagnosis

Estimation of measurement uncertainty of pesticides, polychlorinated biphenyls and polyaromatic hydrocarbons in sediments by using gas chromatography–mass spectrometry by Óscar Pindado Jiménez; Rosa Mª Pérez Pastor (pp. 20-29).

Display Omitted► We report an evaluation of uncertainty associated to analysis of POPs in sediments. ► We give useful and easy to follow guidance to calculate uncertainty. ► Evaluation of uncertainty is essential in environmental analysis. ► Uncertainty derived of calibration is the main contribution. ► We present strategies for diminish uncertainty.The evaluation of the uncertainty associated to analytical methods is essential in order to demonstrate quality of a result. However, there is often lack of information about uncertainty of methods to estimate persistent organic pollutants concentration in complex matrix. Current work has thoroughly evaluated uncertainty associated to quantification of several organochloride pesticides, PCBs and PAHs in sediments. A discussion of the main contributions to the overall uncertainty is reported, allowing authors to establish the accuracy of results and plan future improvements. Combined uncertainties ranged between 5–9% (pesticides), 4–7% (PCBs) and 5–10% (PAHs), being uncertainty derived of calibration the main contribution. Also, the analytical procedure was validated analysing a standard reference material (IAEA-408).

Keywords: Gas chromatography; Pesticide; Polyaromatic hydrocarbons; Polychlorinated biphenyls; Sediments; Uncertainty

Recognition of chemical compounds in contaminated water using time-dependent multiple dose cellular responses by T.H. Pan; B. Huang; J.Z. Xing; W.P. Zhang; S. Gabos; J. Chen (pp. 30-39).

Display Omitted► Dose- and time-dependent cellular responses are used to evaluate the cytotoxicity. ► The CI can reflect the cell number, cell viability, morphological change, etc. ► The CSVID can capture the dynamic information after cells exposed to toxins. ► The multi-class classification can distinguish the compounds using multi-doses. ► The majority vote strategy (fingerprint) can improve the classification accuracy.An early determination of toxicant compounds of water contaminations can gain critical time to protect citizens’ health and save substantial amounts of medical costs. To determine toxins in real time, a multi-dose classification algorithm using cellular state variable identification (CSVID) is developed in this paper. First, the dynamic cytotoxicity response profiles of living cells are measured using a real-time cell electronic sensing (RT-CES) system. Changes in cell number expressed as cell index (CI) are recorded on-line as time series. Then CSVID, which reflects the cell killing, cell lysis and certain cellular pathological changes, is extracted from those dynamic cellular responses. Finally, a support vector machine (SVM) algorithm based on CSVID is employed to classify chemical compounds and determine their analogous cellular response pathway. In order to increase the classification accuracy, a majority vote of the class labels is also proposed. Several validation studies demonstrate that CSVID-based classification algorithm has great potential in distinguishing the cytotoxicity response of the cells in the presence of toxins.

Keywords: Cytotoxicity; Real-time cell electronic sensor system; Water protection; Support vector machine; Multi-class classification; Cellular state variable identification

Study on the application of reduced graphene oxide and multiwall carbon nanotubes hybrid materials for simultaneous determination of catechol, hydroquinone, p-cresol and nitrite by Fangxin Hu; Shihong Chen; Chengyan Wang; Ruo Yuan; Dehua Yuan; Cun Wang (pp. 40-46).

In this paper, the reduced graphene oxide and multiwall carbon nanotubes hybrid materials (RGO–MWNTs) were prepared and a novel strategy for the simultaneous determination of multiple environmental contaminations has been proposed on the basis of RGO–MWNTs hybrid materials modified electrode. The hybrid materials were characterized by the scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and N₂ sorption–desorption isotherms. Due to the excellent catalytic activity, enhanced electrical conductivity, high surface area and porous structure of the RGO–MWNTs, the RGO–MWNTs/GCE achieved the simultaneous measurement of hydroquinone (HQ), catechol (CC), p-cresol (PC) and nitrite (NO₂⁻) with well-separate four peaks. Scheme 1a illuminated the preparation process of the RGO–MWNTs hybrid materials. Scheme 1b explains the electron mediating properties of RGO–MWNTs/GCE towards the oxidation of HQ, CC, PC and NO₂⁻. Scheme 1c presented the SEM image of RGO–MWNTs hybrid materials. Scheme 1d and e showed the 2D and 3D AFM images of RGO–MWNTs films, respectively.Display Omitted► The novel RGO–MWNTs hybrid materials were synthesized. ► The simultaneous detection of four environmental contaminations was achieved. ► SEM, AFM, XPS was employed to characterize the RGO–MWNTs hybrid materials.In this paper, the reduced graphene oxide and multiwall carbon nanotubes hybrid materials (RGO–MWNTs) were prepared and a strategy for detecting environmental contaminations was proposed on the basis of RGO–MWNTs modified electrode. The hybrid materials were characterized by the scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and N₂ sorption–desorption isotherms. Due to the excellent catalytic activity, enhanced electrical conductivity and high surface area of the RGO–MWNTs, the simultaneous measurement of hydroquinone (HQ), catechol (CC), p-cresol (PC) and nitrite (NO₂⁻) with four well-separate peaks was achieved at the RGO–MWNTs modified electrode. The linear response ranges for HQ, CC, PC and NO₂⁻ were 8.0–391.0μM, 5.5–540.0μM, 5.0–430.0μM and 75.0–6060.0μM, correspondingly, and the detection limits (S/N=3) were 2.6μM, 1.8μM, 1.6μM and 25.0μM, respectively. The outstanding film forming ability of RGO–MWNTs hybrid materials endowed the modified electrode enhanced stability. Furthermore, the fabricated sensor was applied for the simultaneous determination of HQ, CC, PC and NO₂⁻ in the river water sample.

Keywords: Reduced graphene oxide; Multiwall carbon nanotubes; Hybrid materials; Environmental contamination; Simultaneous detection

In-syringe demulsified dispersive liquid–liquid microextraction and high performance liquid chromatography–mass spectrometry for the determination of trace fungicides in environmental water samples by Yating Xia; Min Cheng; Feng Guo; Xiangfang Wang; Jing Cheng (pp. 47-53).

Display Omitted► The pretreatment apparatus is the first introduced. ► The pipette tip is a efficient preconcentration tool. ► The LOQ of the proposed technique is very low.An in-syringe demulsified dispersive liquid–liquid microextraction (ISD–DLLME) technique was developed using low-density extraction solvents for the highly sensitive determination of the three trace fungicides (azoxystrobin, diethofencarb and pyrimethanil) in water samples by high performance liquid chromatography–mass spectrometry chromatography–diode array detector/electrospray ionisation mass spectrometry. In the proposed technique, a 5-mL syringe was used as an extraction, separation and preconcentration container. The emulsion was obtained after the mixture of toluene (extraction solvent) and methanol (dispersive solvent) was injected into the aqueous bulk of the syringe. The obtained emulsion cleared into two phases without centrifugation, when an aliquot of methanol was introduced as a demulsifier. The separated floating organic extraction solvent was impelled and collected into a pipette tip fitted to the tip of the syringe. Under the optimal conditions, the enrichment factors for azoxystrobin, diethofencarb and pyrimethanil were 239, 200, 195, respectively. The limits of detection, calculated as three times the signal-to-noise ratio (SN⁻¹), were 0.026μgL⁻¹ for azoxystrobin, 0.071μgL⁻¹ for diethofencarb and 0.040μgL⁻¹ for pyrimethanil. The repeatability study was carried out by extracting the spiked water samples at concentration levels of 0.02μgmL⁻¹ for all the three fungicides. The relative standard deviations varied between 4.9 and 8.2% ( n=5). The recoveries of all the three fungicides from tap, lake and rain water samples at spiking levels of 0.2, 1, 5μgL⁻¹ were in the range of 90.0–105.0%, 86.0–114.0% and 88.6–110.0%, respectively. The proposed ISD–DLLME technique was demonstrated to be simple, practical and efficient for the determination of different kinds of fungicide residues in real water samples.

Keywords: In-syringe; Demulsified dispersive liquid–liquid micoextraction; Fungicide; High performance liquid chromatography–mass spectrometery

Preparation of Fe₃O₄ nanoparticle enclosure hydroxylated multi-walled carbon nanotubes for the determination of aconitines in human serum samples by Hong-Fei Zhang; Yan-Ping Shi (pp. 54-60).

A magnetic carbon nanomaterial of Fe₃O₄ enclosure hydroxylated multi-walled carbon nanotubes (Fe₃O₄-EC-MWCNTs-OH) was prepared by the aggregated effect of Fe₃O₄ nanoparticles on MWCNTs (multi-walled carbon nanotubes)-OH, and combined with high-performance liquid chromatography-diode array detection to determine the aconitines (aconitine, hypaconitine and mesaconitine) in human serum samples. Compared with other extraction modes investigated in experiment, Fe₃O₄-EC-MWCNTs-OH sorbents showed a good affinity to target analytes. Some important parameters that could influence extraction efficiency of aconitines, including the extraction mode, amounts of Fe₃O₄-EC-MWCNTs-OH, pH of sample solution, extraction time, desorption solvent and desorption time, were optimized. Under optimal conditions, the recoveries of spiked serum samples were between 98.0% and 103.0%; relative standard deviations ranged from 0.9% to 6.2%. The correlation coefficients varied from 0.9996 to 0.9998. The limits of detection (LOD) ranged from 3.1ngmL⁻¹ to 4.1ngmL⁻¹ at a signal-to-noise ratio of 3. The experimental results showed that the proposed method was feasible for the analysis of aconitines in serum samples.Display Omitted► The material of Fe₃O₄-enlosure MWCNTs-OH was prepared with chemical co-precipitation method. ► The material synthesized was used as sorbents to extract aconitines from human serum samples. ► Results exhibited the developed method is feasible for analysis in complex matrix.A magnetic carbon nanomaterial for Fe₃O₄ enclosure hydroxylated multi-walled carbon nanotubes (Fe₃O₄-EC-MWCNTs-OH) was prepared by the aggregating effect of Fe₃O₄ nanoparticle on MWCNTs-OH, and combined with high-performance liquid chromatography (HPLC)/diode array detection (DAD) to determine the aconitines (aconitine, hypaconitine and mesaconitine) in human serum samples. Compared with other extraction modes investigated in experiment, Fe₃O₄-EC-MWCNTs-OH sorbents showed a good affinity to target analytes. Some important parameters that could influence extraction efficiency of aconitines, including the extraction mode, amounts of Fe₃O₄-EC-MWCNTs-OH, pH of sample solution, extraction time, desorption solvent and desorption time, were optimized. Under optimal conditions, the recoveries of spiked serum samples were between 98.0% and 103.0%; relative standard deviations (RSDs) ranged from 0.9% to 6.2%. The correlation coefficients varied from 0.9996 to 0.9998. The limits of detection ranged from 3.1ngmL⁻¹ to 4.1ngmL⁻¹ at a signal-to-noise ratio of 3. The experimental results showed that the proposed method was feasible for the analysis of aconitines in serum samples.

Keywords: Fe; ₃; O; ₄; nanoparticles; Hydroxylated multi-walled carbon nanotubes; Aconitines; Serum samples

A novel sorptive extraction method based on polydimethylsiloxane frit for determination of lung cancer biomarkers in human serum by Hui Xu; Shuyu Wang (pp. 61-66).

Display Omitted► Porous polypropylene frit was used as a support of PDMS coating. ► Method was established for analysis of aldehydes biomarkers in human serum samples. ► Compared with SBSE, the developed method is simple, labor-saving, rapid, robust and sensitive. ► It enables effective cleanup of sample matrix simultaneously with enrichment of target analytes.In this study, a porous polypropylene frit was coated with polydimethylsiloxane (PDMS) as extraction medium, based on the home-made PDMS-frit, a rapid, simple and sensitive sorptive extraction method was established for analysis of potential biomarkers of lung cancer (hexanal and heptanal) in human serum samples. In the method, derivatization and extraction occurred simultaneously on the PDMS-frit, then the loaded frit was ultrasonically desorbed in acetonitrile. Polymerization, derivatization–extraction and desorption conditions were optimized. Under the optimal conditions, satisfactory results were gained, a wide linear application range was obtained in the range of 0.002–5.0μmolL⁻¹ ( R>0.997) for two aldehydes, the detection limits ( S N⁻¹=3) were 0.5nmolL⁻¹ for hexanal and 0.4nmolL⁻¹ for heptanal. The relative standard deviations (RSDs, n=5) of the method were below 7.9% and the recoveries were above 72.7% for the spiked serum. All these results hint that the proposed method is potential for disease markers analysis in complex biological samples.

Keywords: High performance liquid chromatography (HPLC); Polydimethylsiloxane (PDMS); Frit; Aldehyde; Serum analysisAbbreviations; HPLC; high-performance liquid chromatography; PDMS; polydimethylsiloxane; DNPH; 2,4-dinitrophenylhydrazine; SPME; solid-phase microextraction; SBSE; stir bar sorptive extraction

A simple, reproducible and sensitive spectrophotometric method to estimate microalgal lipids by Yimin Chen; Seetharaman Vaidyanathan (pp. 67-72).

Display Omitted► FAs released from lipids form complex with Cu–TEA in chloroform. ► The FA–Cu–TEA complex gives strong absorbance at 260nm. ► The absorbance is sensitive and independent of C-atom number in the FAs (10–18). ► Microalgal lipid extract and pure FA (such as C16) can both be used as standards.Quantification of total lipids is a necessity for any study of lipid production by microalgae, especially given the current interest in microalgal carbon capture and biofuels. In this study, we employed a simple yet sensitive method to indirectly measure the lipids in microalgae by measuring the fatty acids (FA) after saponification. The fatty acids were reacted with triethanolamine–copper salts (TEA–Cu) and the ternary TEA–Cu–FA complex was detected at 260nm using a UV–visible spectrometer without any colour developer. The results showed that this method could be used to analyse low levels of lipids in the range of nano-moles from as little as 1mL of microalgal culture. Furthermore, the structure of the TEA–Cu–FA complex and related reaction process are proposed to better understand this assay. There is no special instrument required and the method is very reproducible. To the best of our knowledge, this is the first report of the use of UV absorbance of copper salts with FA as a method to estimate lipids in algal cultures. It will pave the way for a more convenient assay of lipids in microalgae and can readily be expanded for estimating lipids in other biological systems.

Keywords: Lipids; Biofuel; Microalgae; Spectrophotometry; Fatty acids

Desalting of phosphopeptides by tandem polypyrrole-c18 reverse phase micropipette tip (TMTip_PPY-C18) based on hybrid electrostatic, Π–Π stacking and hydrophobic interactions for mass spectrometric analysis by Shi Zheng; Xiaoli Wang; Jieying Fu; Xuejiao Hu; Xiao Xiao; Lulu Huang; Youe Zhou; Hongying Zhong (pp. 73-79).

Display Omitted► A new micropipette tip TMTip_PPY-C18 was developed for desalting of phosphopeptides. ► TMTip_PPY-C18 is based on polypyrrole in tandem with C18 chromatographic material. ► TMTip_PPY-C18 combines electrostatic, Π–Π stacking and hydrophobic interactions. ► TMTip_PPY-C18 can be used in both acidic and basic experimental conditions.Desalting and concentration of peptides using reverse phase (RP) C18 chromatographic material based on hydrophobic interaction is a routine approach used in mass spectrometry (MS)-based proteomics. However, MS detection of small hydrophilic peptides, in particular, phosphopeptides that bear multiple negative charges, is challenging due to the insufficient binding to C18 stationary phase. We described here the development of a new desalting method that takes the unique properties of polypyrrole (PPY). The presence of positively charged nitrogen atoms under acidic conditions and polyunsaturated bonds in polypyrrole provide a prospect for enhanced adsorption of phosphopeptides or hydrophilic peptides through extra electrostatic and Π–Π stacking interactions in addition to hydrophobic interactions. In tandem with reversed phase C18 chromatographic material, the new type of desalting method termed as TMTip_PPY-C18 can significantly improve the MS detection of phosphopeptides with multiple phosphate groups and other small hydrophilic peptides. It has been applied to not only tryptic digest of model proteins but also the analysis of complex lysates of zebrafish eggs. The number of detected phosphate groups on a peptide ranged from 1 to 6. Particularly, polypyrrole based method can also be used in basic condition. Thus it provides a useful means to handle peptides that may not be detectable in acidic condition. It can be envisioned that the TMTip_PPY-C18 should be able to facilitate the exploration of large scale phosphoproteome.

Keywords: Electrostatic interaction; Π–Π stacking interaction; Hydrophobic interaction; Phosphopeptides; Desalting; Mass spectrometry

Colorimetric detection of cholesterol with G-quadruplex-based DNAzymes and ABTS²⁻ by Ruimin Li; Cen Xiong; Zhiyou Xiao; Liansheng Ling (pp. 80-85).

A novel colorimetric method for detection of cholesterol was developed with hemin-G-quadruplex DNAzyme by transducing oxidation of cholesterol into the color change of ABTS²⁻.Display Omitted► A novel colorimetric method for detection of cholesterol was developed with hemin-G-quadruplex DNAzyme and ABTS² ► The detection limit of the assay was 0.1μM. ► Satisfactory results were obtained when the assay was applied in the detection of cholesterol in human serum.A novel colorimetric method for detection of cholesterol was developed with hemin-G-quadruplex DNAzyme by transducing oxidation of cholesterol into the color change of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS²⁻). Oligonucleotide 5′-GTGGGTAGGGCGGGTTGG-3′ (Oligo-1) formed G-quadruplex structure in the presence of K⁺, it acted as a horseradish peroxidase (HRP) mimicking DNAzyme when binding hemin and catalyzed the oxidation of colorless ABTS²⁻ to green ABTS⁻ by H₂O₂, which was produced by the reaction of cholesterol and oxygen that catalyzed by cholesterol oxidase. Therefore, the oxidation of cholesterol could be transduced into the color change of ABTS²⁻ by combining these two reactions. Under the optimum conditions, the absorbance was proportional to the concentration of cholesterol over the range of 1.0–30μM, with a linear regression equation of A=0.362+0.0256 C ( C: μM, R=0.998) and a detection limit of 0.10μM (3 σ/slope). Moreover, the practicability of the assay in the detection of cholesterol in human serum was studied as well.

Keywords: Cholesterol; Colorimetric detection; DNAzyme; ABTS; ²⁻

Gap site-specific rapid formation of fluorescent silver nanoclusters for label-free DNA nucleobase recognition by Qinghua Cui; Kun Ma; Yong Shao; Shujuan Xu; Fei Wu; Guiying Liu; Norio Teramae; Haifeng Bao (pp. 86-91).

Display Omitted► A gap site in DNA duplex is exploited as a new scaffold for the rapid synthesis of Ag NCs. ► Fluorescent Ag NCs are highly selectively formed when cytosine faced toward the gap site in DNA duplexes, and they could be utilized as readout with signal-on signaling for DNA single nucleotide polymorphism. ► This base-selective growth of the fluorescent Ag NCs at the gap site would find promising applications in label-free and cost-effective designs of DNA-based functional sensors.Silver nanoclusters (Ag NCs) templated with DNAs have attracted much attention as novel fluorophores because of their convenient emission tunability by the sequence and length of the template DNAs. However, the precise production of Ag NCs in a site-specific manner still remains a challenge to attain highly selective and label-free DNA recognition. Here we exploited the availability of a gap site in DNA duplexes as a new scaffold for the synthesis of Ag NCs. Compared to the commonly used DNA templates for the creation of Ag NCs, the gap site in DNA duplexes was found to facilitate the rapid formation of the fluorescent Ag NCs without sacrifice of their bright emission and excellent stability. We found that fluorescent Ag NCs were highly selectively formed when cytosine faced toward the gap site in DNA duplexes, and they were in situ utilized as readout by signal-on manner for the DNA mutation assays. This base-selective growth of the fluorescent Ag NCs at the gap site would find promising applications in practical detection of single nucleotide polymorphism (SNP) and construction of DNA-based functional sensors with label-free and cost-effective merits.

Keywords: Silver nanoclusters; DNA; Nucleobase recognition; Gap site; Fluorescence

Development of a novel label-free amperometric immunosensor for the detection of okadaic acid by Akhtar Hayat; Lise Barthelmebs; Audrey Sassolas; Jean-Louis Marty (pp. 92-97).

Display Omitted► This work described a new approach for label-free amperometric detection of OA. ► The use of protein G MBs provided large surface area for immunochemical reaction. ► The specific interaction between immobilized anti-OA-MAb and OA increased R_et without any amplification step. ► DPV was carried out to perform a calibration curve.Okadaic acid (OA), a lipophilic phycotoxin is mainly produced by toxigenic dinoflagellates. The need to develop high performing methods for OA analysis able to improve the traditional ones is evident. In this work, a novel experimental methodology for label-free detection of OA was developed. Protein G magnetic beads (protein-G-MBs) modified gold electrode was used to immobilize anti-OA monoclonal antibody (anti-OA-MAb). Preliminary, colorimetric tests were performed in order to validate protein-G-MBs and anti-OA-MAb reaction. Electrochemical detection was carried out by differential pulse voltammetry in ferri/ferrocyanide solution. The limit of detection value obtained (0.5μgL⁻¹) validated the developed electrochemical immunosensor as a promising tool for routine use. The matrix effect and the recovery rate were also assessed with real samples, showing a good percentage of recovery.

Keywords: Okadaic acid; Protein G magnetic beads; Label-free immunosensor; Amperometric immunosensor; Mussel extract

Development of a multi-component chemically reactive detection conjugate for the determination of Hg(II) in water samples by Yu Zhou; Xiang-Li Tian; Yan-Song Li; Yuan-Yuan Zhang; Zhao-Hui Li; Li Yang; Jun-Hui Zhang; Xin-Rui Wang; Xian-Mei Meng; Jing-Qiu Liu (pp. 98-103).

Display Omitted► A multi-component probe for the determination of Hg(II) was synthesized. ► A platform based on the multi-component probe was proposed and applied for Hg(II) detection in water samples. ► The application results obtained from the developed technique has a close correlation with those from ICP-MS.Mercury ions (Hg(II)) are considered highly toxic and hazardous element even at low levels. The contamination of Hg(II) is a global problem. To develop selective and sensitive technique for the detection of Hg(II) has attracted considerable attention. In this study, a multi-component chemically reactive detection conjugate for determination of Hg(II) has been synthesized and a competitive format assay was proposed. In the technique, the chemically reactive capture conjugate was coated on the plate. The reactive detection conjugate was then captured by the capture conjugate. TMB solution was added and catalyzed by HRP molecules immobilized on AuNPs. Finally, the developed enzymatic signal was measured at 450nm. The linear range of the assay was 0.35–350ppb with a detection limit of 0.1ppb. The average recoveries of Hg(II) from mineral water, tap water and lake water were 100.03%, 103.13% and 102.03%, respectively. All coefficients of variation (CVs) were less than 10%. The results are closely correlated with those from inductively coupled plasma mass spectrometry (ICP-MS), which indicated that the developed technique is a reliable method for and sensitive detection of Hg(II) in water samples.

Keywords: Hg(II); Gold nanoparticles; Horseradish peroxidase; Detection

Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease by Fengyun Li; Junping Yu; Zhiping Zhang; Zongqiang Cui; Dianbing Wang; Hongping Wei; Xian-En Zhang (pp. 104-110).

.Display Omitted► We studied some buffer factors affecting BRET ratio for the first time. ► hGluc and EYFP were genetically linked by protease cleavable fragments. ► An optimized buffer improved 10-fold sensitivity, 7-fold limit of protease detection. ► The enhancing buffer optimized the protease reaction and the BRET simultaneously.Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.

Keywords: Bioluminescence resonance energy transfer; Gaussia; luciferase; Enhanced yellow fluorescence protein; Enterokinase

Sequential-dissociation kinetics of non-covalent complexes of DNA with multiple proteins in separation-based approach: General theory and its application by Leonid T. Cherney; Victor Okhonin; Alexander P. Petrov; Sergey N. Krylov (pp. 111-118).

Display Omitted► Separation-based approach facilitates studying kinetics of protein assembly/disassembly on DNA. ► Solution was obtained to find kinetic constants for sequential disassembly of proteins from DNA. ► The found solution can be applied to other “sequential decay” processes.Binding of multiple proteins to DNA is crucial in many regulatory cellular processes. The kinetics of assembly and disassembly of DNA–multiple protein complexes is very difficult to study in detail due to the lack of suitable experimental approaches. A separation-based approach has been recently proposed to resolve disassembly kinetics of such complexes. While conceptually simple, the separation-based approach generates experimental data with very complex patterns. The analysis of these patterns is a challenging problem on its own. Here we report on a mathematical approach that can extract a solution for the experimental data obtained in separation-based analysis of sequential dissociation of a DNA complex with multiple proteins. This case describes the dissociation of proteins one-by-one from the complex. Generally speaking, a mathematical solution of such problems requires calculations of multiple integrals. Our approach reduces this procedure to taking double integrals and constructing their superposition. We tested this approach with the experimental data obtained for three-step sequential dissociation of complexes of DNA with two protein copies.

Keywords: Abbreviations; CE; capillary electrophoresis; SPR; surface plasmon resonance; SSB; single strand DNA bindingProtein–DNA interaction; Protein machines; Kinetics; Capillary electrophoresis

A selective metabolite array for the detection of phosphometabolites by Malinda Salim; Gregory J.S. Fowler; Phillip C. Wright; Seetharaman Vaidyanathan (pp. 119-126).

Display Omitted► Metal affinity based selective array for the screening of phosphometabolites. ► A simple one-step plasma polymer coating and direct metal immobilisation. ► ToF-SIMS detection of phosphometabolite array. ► Ga and Zr suitable metals.Immobilised metal ion affinity (IMA) has been traditionally used specifically for the separation of phosphorylated proteins and nucleic acids, in proteomics and genomics, respectively. This report describes the novel application of IMA in metabolomics for the development of metabolite arrays to detect phosphometabolites using a plasma polymer-modified surface. Immobilisation of gallium, zirconium, cobalt, copper, zinc, nickel, iron, and chromium to acrylic acid plasma polymer followed by subsequent exposure to metabolites (phospho- and non-phosphometabolites) was investigated. Results analysed using ToF-SIMS suggests that gallium and zirconium exhibit higher phosphometabolite affinity and specificity compared to other metals, and can be used to develop metabolite arrays for the detection of phosphometabolites.

Keywords: Metal affinity; Metabolomics; Phosphate; Secondary ion mass spectrometry; Metabolite profiling

Simultaneous qualitative assessment and quantitative analysis of flavonoids in various tissues of lotus ( Nelumbo nucifera) using high performance liquid chromatography coupled with triple quad mass spectrometry by Sha Chen; Linchuan Fang; Huifen Xi; Le Guan; Jinbao Fang; Yanling Liu; Benhong Wu; Shaohua Li (pp. 127-135).

Mass spectra model and structures of 20 flavonoids found in various tissues of lotus in this study.Display Omitted► Simultaneous flavonoids separation protocols were established for various lotus ( Nelumbo nucifera) tissues. ► Twenty flavonoids were identified and four were found for the first time in N. nucifera. ► Flavonoid compositions and contents greatly varied with lotus tissues.Flavonoid composition and concentration were investigated in 12 different tissues of ‘Ti-1’ lotus ( Nelumbo nucifera) by high performance liquid chromatography equipped with photodiode array detection tandem electrospray ionization mass spectrometry (HPLC-DAD-ESI-MSⁿ). A total of 20 flavonoids belonging to six groups (myricetin, quercetin, kaempferol, isohamnetin, diosmetin derivatives) were separated and identified. Myricetin 3- O-galactoside, myricetin 3- O-glucuronide, isorhamnetin 3- O-glucuronide and free aglycone diometin (3′,5,7-trihydroxy-4′-methoxyflavone) were first reported in lotus. Flavonoid composition varied largely with tissue type, and diverse compounds (5–15) were found in leaf and flower stalks, flower pistils, seed coats and embryos. Flower tissues including flower petals, stamens, pistils, and, especially, reproductive tissue fruit coats had more flavonoid compounds (15–17) than leaves (12), while no flavonoids were detectable in seed kernels. The flavonoid content of seed embryos was high, 730.95mg 100g⁻¹ DW (dry weight). As regards the other tissues, mature leaf pulp (771.79mg 100g⁻¹ FW (fresh weight)) and young leaves (650.67mg 100g⁻¹ FW) had higher total flavonoid amount than flower stamens (359.45mg 100g⁻¹ FW) and flower petals (342.97mg 100g⁻¹ FW), while leaf stalks, flower stalks and seed coats had much less total flavonoid (less than 40mg 100g⁻¹ FW).

Keywords: HPLC; Mass spectrometry; Flavonoids; Lotus; Tissues

Poly (acrylic acid) microchannel modification for the enhanced resolution of catecholamines microchip electrophoresis with electrochemical detection by Isabel Álvarez-Martos; M. Teresa Fernández-Abedul; Adela Anillo; José Luis G. Fierro; Francisco Javier García Alonso; Agustín Costa-García (pp. 136-143).

Display Omitted► PAA-glass is made by activation, silanization and immobilization on an amine surface. ► PAA-attachment to a glass microchannel improves the resolution of neurotransmitters. ► Methanol addition and microchannel length further enhance separation of PAA-devices.A new modification of glass electrophoresis microchips based on poly (acrylic) acid immobilization has been performed. It is based on the reaction of PAA with an amine functionalized surface, obtained through the bifunctional reagent 3-aminopropyl triethoxysilane. Parameters affecting all the three steps involved: surface activation, silanization and polymer immobilization were optimized employing soda-lime glass plates. Characterization by SEM and XPS was carried out. Application of the modified microchips to the separation of a model system: dopamine (D), epinephrine (E) and norepinephrine (NE), that on the other hand are of high clinical relevance was performed employing amperometric detection. Modification is necessary for obtaining partial resolution of all the three analytes in a microchip with an effective separation length of 30mm. Situation changes from no resolution (Rs) at all (only one peak was achieved for the mixture) to a partial resolution (Rs D–NE and Rs NE–E are 0.25 and 0.24 respectively). Microchips with 60mm of separation channel were also modified, implying this procedure a resolution enhancement (Rs of 0.49 and 0.28 for D–NE and NE–E respectively), even when methanol is employed as organic modifier (Rs values of 0.70 (D–NE) and 0.66 (NE–E) for a 3% MeOH).

Keywords: Microchip electrophoresis; Gold end-channel amperometric detection; Surface modification; Poly (acrylic acid) polymer; Catecholamines

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Analytica Chimica Acta (v.724, #)