Analytica Chimica Acta (v.964, #C)

A new technology to quantify phospho-p5315 by converting its content to the amount of glucose which is detectable by a glucometer was developed. An immunochromatographic test strip (ITS) was used as a disposable platform, where primary antibody (Ab1)-modified Fe3O4 magnetic nanoparticles (Fe3O4-Ab1) were settled on the test zone to capture both the target phospho-p5315 and the detection antibody (Ab2)-glucose encapsulating liposome (GEL) conjugate. The measurement was based on the release and subsequent detection of glucose from Ab2-GEL using a glucose meter (GM). The amount of glucose is proportional to the phospho-p5315 concentration from 0.1 to 50 ng mL−1, the limit of detection is 50 pg mL−1 (3S/N). The high sensitivity was a result of the huge number of glucose encapsulated in the liposome. Taking the advantage of low cost, widespread availability and portability of the test trip, together with the personal GM, the presented approach can be easily used to detect other disease biomarkers in medical diagnostics and environmental monitoring.A new technology to quantify phospho-p5315 by converting its content to the amount of glucose which is detectable by a glucometer was developed. An immunochromatographic test strip (ITS) was used as a disposable platform. The measurement was based on the release and subsequent detection of glucose from Ab2-GEL using a glucose meter (GM). Taking the advantage of low cost, widespread availability and portability of the test trip, together with the personal GM, the presented approach can be easily used to detect other disease biomarkers in medical diagnostics and environmental monitoring.Display Omitted
Keywords: Immunochromatographic test strip; Glucometer; Liposome; Phospho-p5315;

Mass spectrometry (MS) based proteomics have achieved a near-complete proteome coverage in humans and in several other organisms, producing a wealth of information stored in databases and bioinformatics resources. Recent implementation of selected/multiple reaction monitoring (SRM/MRM) technology in targeted proteomics introduced the possibility of quantitatively follow-up specific protein targets in a hypothesis-driven experiment. In contrast to immunoaffinity-based workflows typically used in biological and clinical research for protein quantification, SRM/MRM is characterized by high selectivity, large capacity for multiplexing (approx. 200 proteins per analysis) and rapid, cost-effective transition from assay development to deployment. The concept of SRM/MRM utilizes triple quadrupole (QqQ) mass analyzer to provide inherent reproducibility, unparalleled sensitivity and selectivity to efficiently differentiate isoforms, post-translational modifications and mutated forms of proteins. SRM-like targeted acquisitions such as parallel reaction monitoring (PRM) are pioneered on high resolution/accurate mass (HR/AM) platforms based on the quadrupole-orbitrap (Q-orbitrap) mass spectrometer. The expansion of HR/AM also caused development in data independent acquisition (DIA). This review presents a step-by-step tutorial on development of SRM/MRM protein assay intended for researchers without prior experience in proteomics. We discus practical aspects of SRM-based quantitative proteomics workflow, summarize milestones in basic biological and medical research as well as recent trends and emerging techniques.Display Omitted
Keywords: Targeted quantitative proteomics tutorial; Selected/multiple reaction monitoring (SRM/MRM); Parallel reaction monitoring (PRM); Tandem mass spectrometry (MS/MS); Data independent acquisition (DIA); Sequential windowed acquisition of all theoretical fragmentation spectra (SWATH);

Porous monoliths for on-line sample preparation: A review by Jorge C. Masini; Frantisek Svec (24-44).
This review aims at presenting the state of the art concerning monolithic materials for on-line sample preparation emphasizing solid-phase extraction, matrix exchange, and analyte conversion. Emphasis was given to organic and silica-based, as well as hybrid monoliths reported in the literature mostly after 2010. The first part of this review presents materials and strategies for enrichment of inorganic species in environmental and biological samples using mostly ICP-MS detectors. In the second part we focus on organic analytes, discussing the role of surface area of the polymer monoliths and density of adsorption sites for specific interactions, including incorporation of nanoparticles, metal organic frameworks, as well as the preparation of hybrid organic-silica monoliths to increase the surface area. Incorporation of ionic liquids to increase the number of types of interaction mechanisms available for retention is also discussed. Monoliths affording molecular recognition properties achieved by including boronate moieties for cis-diol recognition, as well as antibodies and aptamers for specific molecular recognition are also reviewed. The largest number of applications of molecular recognition mechanisms was observed for molecularly imprinted polymer monoliths as a consequence of the simplicity of this approach when compared to the use of immunosorbents or aptamers. The final part examines the on-line applications of immobilized enzyme reactors used for protein digestion in proteomic analysis and for kinetic studies in drug discovery and clinical assays usually coupling the reactors to mass spectrometers.Display Omitted
Keywords: Immobilized enzyme reactors; Liquid chromatography; Molecular recognition; Monolithic columns; Separations; Solid-phase extraction;

Evaluation of ensemble Monte Carlo variable selection for identification of metabolite markers on NMR data by C.A. Esquerre; A.A. Gowen; A. O'Gorman; G. Downey; C.P. O'Donnell (45-54).
The aim of this study was to investigate the potential of the recently developed ensemble Monte Carlo Variable Selection (EMCVS) method to identify the relevant portions of high resolution 1H NMR spectra as a metabolite fingerprinting tool and compare to a widely used method (Variable importance on projection (VIP)) and recently proposed variable selected methods i.e. selectivity ratio (SR) and significance multivariate correlation (sMC). As case studies two quantitative publicly available datasets: wine samples, urine samples of rats, and an experiment on mushroom (Agaricus bisporus) were examined. EMCVS outperformed the three other variable selection methods in most cases, selecting fewer chemical shifts and leading to improved classification of mushrooms and prediction of onion by-products intake and wine components. These fewer chemical shift regions facilitate the interpretation of the NMR spectra, fingerprinting and identification of metabolite markers.Display Omitted
Keywords: NMR; PLS; Variable selection; Enhanced Monte Carlo variable selection;

In this article, we propose the use of the Multivariate Curve Resolution – Alternating Least Squares (MCR-ALS) chemometrics method to resolve the 1H NMR spectra and concentration of the individual metabolites in their mixtures in untargeted metabolomics studies. A decision tree-based strategy is presented to optimally select and implement spectra estimates and equality constraints during MCR-ALS optimization.The proposed method has been satisfactorily evaluated using different 1H NMR metabolomics datasets. In a first study, 1H NMR spectra of the metabolites in a simulated mixture were successfully recovered and assigned. In a second study, more than 30 metabolites were characterized and quantified from an experimental unknown mixture analyzed by 1H NMR. In this work, MCR-ALS is shown to be a convenient tool for metabolite investigation and sample screening using 1H NMR, and it opens a new path for performing metabolomics studies with this chemometric technique.Display Omitted
Keywords: Metabolomics; Nuclear magnetic resonance; Multivariate curve resolution;

Methods based on nanostructures have been developed for potentiometric immunosensors, but most involve low sensitivity or weak signal output and are unsuitable for routine use in diagnosis. Herein, we devise an in-situ signal-amplification strategy for enhanced electrical readout of potentiometric immunosensor toward target prostate-specific antigen (PSA, one kind of cancer biomarkers), based on polyion oligonucleotide-labeled gold nanoparticles (AuNPs). To decrease the background signal, monoclonal anti-human PSA capture antibody was covalently conjugated onto an activated glassy carbon electrode via typical carbodiimide coupling. AuNPs heavily functionalized with the polyion oligonucleotides and polyclonal anti-PSA detection antibodies (pAb2-AuNP-DNA) were utilized as the signal-generation nanotags. In the presence of target PSA, a sandwich-type immunoreaction was executed between capture antibody and detection antibody on the electrode. The detectable signal derived from the shift in the electric potential as a result of the change in the surface charge before and after the antigen-antibody reaction. With target PSA increased, the captured pAb2-AuNP-DNA to the electrode accompanying detection antibody increased, thereby resulting in the change of the electrode potential. Due to numerous polyion oligonucleotides with the negative charge, the signal readout amplified. Under the optimal conditions, the shift in the output potential was proportional to the logarithm of target PSA concentration and displayed a dynamic linear range from 0.05 to 20 ng mL−1 with a detection limit of 13.6 pg mL−1. An intermediate precision of ≤13.2% was accomplished with the batch-to-batch identification. The selectivity was acceptable. The method accuracy was evaluated for human serum specimens, and gave the consistent results between the potentiometric immunosensor and the referenced enzyme-linked immunosorbent assay (ELISA).Display Omitted
Keywords: Potentiometric immunosensor; Polyion oligonucleotides; Gold nanoparticles; Prostate-specific antigen; Signal-amplification strategy;

Inter-laboratory validation of a thin film microextraction technique for determination of pesticides in surface water samples by Hamed Piri-Moghadam; Emanuela Gionfriddo; Angel Rodriguez-Lafuente; Jonathan J. Grandy; Heather L. Lord; Terry Obal; Janusz Pawliszyn (74-84).
The primary goal of the present study is the inter-laboratory evaluation of a thin film microextraction (TFME) technique to be used as an alternative approach to liquid-liquid extraction (LLE). Polydimethylsiloxane/divinylbenzene (PDMS/DVB) and PDMS/DVB-carbon mesh supported membranes were used for the extraction of 23 targeted pesticides, while a thermal desorption unit (TDU) was employed to transfer these analytes to a GC/MS instrument for separation and detection. After optimization of the most critical parameters, both membranes were capable of achieving limits of detection (LOD) in the low ng L−1 range while demonstrating excellent robustness, withstanding up to 100 extractions/desorption cycles. Furthermore, limits of quantification (LOQ) between 0.025 and 0.50 μg L−1 were achieved for the 23 compounds selected from several classes of pesticides with a wide range of polarities. A wide linear range of 0.025–10.0 μg L−1 with strong correlation to response (R2 > 0.99) was attained for most of the studied analytes. Both membranes showed good accuracy and repeatability at three levels of concentration. Moreover, the method was also validated through blind split analyses of 18 surface water samples, collected within 3 months, using TFME at the University of Waterloo and LLE at Maxxam Analytics (Mississauga, ON) which is an accredited commercial analytical laboratory. Good agreement between the two methods was achieved with accuracy values ranging from 70 to 130%, for the majority of analytes in the samples collected. At the concentration levels investigated, 90% of the analytes were quantifiable by TFME, whereas only 53% of the compounds were reportable using the LLE method particularly at concentrations lower than 1 μg L−1. The comparison of TFME and LLE from several analytical aspects demonstrated that the novel TFME method gave similar accuracy to LLE, while providing additional advantages including higher sensitivity, lower sample volume, thus reduced waste production, and faster analytical throughput. Given the sensitivity, simplicity, low cost, accuracy, greenness and relatively fast procedure of TFME, it shows great potential for adoption in analytical laboratories as an alternative to LLE.Display Omitted
Keywords: Thin film microextraction; Liquid liquid extraction; Pesticides; Surface water; Inter-laboratory validation; Trace analysis;

Chemically modified halloysite nanotubes as a solid–phase microextraction coating by Mohammad Saraji; Mohammad Taghi Jafari; Mehdi Mossaddegh (85-95).
Halloysite nanotubes were modified in three simple steps including etching, hydroxylation and amino grafting. The sol–gel technique was used for the chemical bonding of the modified halloysite nanotubes (MHNTs) to fused–silica support. The MHNTs, as a novel adsorbent was applied as a SPME coating. Diazinon, parathion and fenthion were selected as the model compounds to study the extraction efficiency of the coating. Gas chromatography–corona discharge ion mobility spectrometry was applied for the analysis of the extracted analytes. The parameters influencing the extraction efficiency of the method, such as stirring rate, salt effect, extraction temperature and time were optimized. The results showed that the MHNTs fiber had better extraction efficiency than the commercial SPME (PA, PDMS, and PDMS–DVB), bare silica, silica–based HNTs and HNTs–titanium dioxide fibers. The limits of detection were found to be in the range of 0.01–0.03 μg L−1. The limits of quantification were in the range of 0.03–0.07 μg L−1. Also, a good linearity in the range of 0.03–3.0, 0.07–2.0 and 0.03–3.0 μg L−1, was found for diazinon, fenthion and parathion, respectively. The method precision was lower than 7.0 and 8.7% as the intra- and inter-day relative standard deviations, respectively. Agricultural wastewater, cucumber and apple were chosen as the real samples. The spiking recovery values were between 84 (±9) and 97% (±6). The results showed that the method was applicable and suitable for real samples analysis.Display Omitted
Keywords: Modified halloysite nanotubes; Solid-phase microextraction; Gas chromatography–ion mobility spectrometry; Wastewater and fruit samples;

Principles of sol-gel chemistry were utilized to create silica- and germania-based dual-ligand surface-bonded sol-gel coatings providing enhanced performance in capillary microextraction (CME) through a combination of ligand superhydrophobicity and π-π interaction. These organic-inorganic hybrid coatings were prepared using sol-gel precursors with bonded perfluorododecyl (PF-C12) and phenethyl (PhE) ligands. Here, the ability of the PF-C12 ligand to provide enhanced hydrophobic interaction was advantageously combined with π-π interaction capability of the PhE moiety to attain the desired sorbent performance in CME. The effect of the inorganic sorbent component on microextraction performance of was explored by comparing microextraction characteristics of silica- and germania-based sol-gel sorbents. The germania–based dual-ligand sol-gel sorbent demonstrated superior CME performance compared to its silica-based counterpart. Thermogravimetric analysis (TGA) of the created silica- and germania-based dual-ligand sol-gel sorbents suggested higher carbon loading on the germania-based sorbent. This might be indicative of more effective condensation of the organic ligand-bearing sol-gel-active chemical species to the germania-based sol-gel network (than to its silica-based counterpart) evolving in the sol solution. The type and concentration of the organic ligands were varied in the sol-gel sorbents to fine-tune extraction selectivity toward different classes of analytes. Specific extraction (SE) values were used for an objective comparison of the prepared sol-gel CME sorbents. The sorbents with higher content of PF-C12 showed remarkable affinity for aliphatic hydrocarbons. Compared to their single-ligand sol-gel counterparts, the dual-ligand sol-gel coatings demonstrated significantly superior CME performance in the extraction of alkylbenzenes, providing up to ∼65.0% higher SE values. The prepared sol-gel CME coatings provided low ng L−1 limit of detections (LOD) (4.2–26.3 ng L−1) for environmentally important analytes including polycyclic aromatic hydrocarbons, ketones and aliphatic hydrocarbons. In CME-GC experiments (n = 5), the capillary-to-capillary RSD value was ∼2.1%; such a low RSD value is indicative of excellent reproducibility of the sol-gel method used for the preparation of these CME coatings. The dual-ligand sol-gel coating provided stable performance in capillary microextraction of analytes from saline samples.Display Omitted
Keywords: Sol-gel coatings for capillary microextraction (CME); Germania-based microextraction media; Perfluorinated alkyl ligands providing superhydrophobicity; Aromatic CME ligands for π-π interaction; Specific extraction (SE); Saline sample matrix;

Non-targeted LC-MS based metabolomics analysis of the urinary steroidal profile by Amelia Palermo; Francesco Botrè; Xavier de la Torre; Nicola Zamboni (112-122).
The urinary steroidal fraction has been extensively explored as non-invasive alternative to monitor pathological conditions as well as to unveil the illicit intake of pseudo-endogenous anabolic steroids in sport. However, the majority of previous approaches involved the a priori selection of potentially relevant target analytes. Here we describe the non-targeted analysis of the urinary steroidal profiles. The workflow includes minimal sample pretreatment and normalization according to the specific gravity of urine, a 20 min reverse phase ultra-performance liquid chromatographic separation hyphenated to electrospray time-of-flight mass spectrometry. As initial validation, we analyzed a set of quality control urines spiked with glucurono- and sulfo-conjugated steroids at physiological ranges. We then applied the method for the analysis of samples collected after single transdermal administration of testosterone in hypogonadal men. The method allowed profiling of approximately three thousand metabolic features, including steroids of clinical and forensic relevance. It successfully identified metabolic pathways mostly responsible for groups clustering even in the context of high inter-individual variability and allowed the detection of currently unknown metabolic features correlating with testosterone administration. These outcomes set the stage for future studies aimed at implementing currently monitored urinary steroidal markers both in clinical and forensic analysis.Display Omitted
Keywords: Urinary steroidal profile; Non-targeted metabolomics; Anabolic androgenic steroids; Testosterone gel; Glucocorticoids; Liquid chromatography; Mass spectrometry;

In this study, gas chromatography (GC) was interfaced with high resolution mass spectrometry (HRMS) with electrospray ionization source (ESI) and the relevant parameters were investigated to enhance the ionization efficiency. In GC-ESI, the distances (x-, y- and z) and angle between the ESI needle, GC capillary column and MS orifice were set to 7 (x-distance), 4 (y-distance), and 1 mm (z-distance). The ESI spray solvent, acid modifier and nebulizer gas flow were methanol, 0.1% formic acid and 5 arbitrary units, respectively. Based on these results, analytical conditions for GC-ESI/HRMS were established. In particular, the results of spray solvent flow indicated a concentration-dependent mechanism (peak dilution effect), and other parameters also greatly influenced the ionization performance. The developed GC-ESI/HRMS was then applied to the analysis of anabolic steroids as trimethylsilyl (TMS) derivatives in human urine to demonstrate its application. The ionization profiles of TMS-derivatized steroids were investigated and compared with those of underivatized steroids obtained from gas chromatography-electrospray ionization/mass spectrometry (GC-ESI/MS) and liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI/MS). The steroids exhibited ionization profiles based on their structural characteristics, regardless of the analyte phase or derivatization. Groups I and II with conjugated or unconjugated keto functional groups at C3 generated the [M+H]+ and [M+H-TMS]+ ions, respectively. On the other hand, Groups III and IV gave rise to the characteristic fragment ions [M+H-TMS-H2O]+ and [M+H-2TMS-H2O]+, corresponding to loss of a neutral TMS·H2O moiety from the protonated molecular ion by in-source dissociation. To the best of our knowledge, this is the first study to successfully ionize and analyze steroids as TMS derivatives using ESI coupled with GC. The present system has enabled the ionization of TMS derivatives under ESI conditions and this method has potential as a novel ionization tool. It is also useful for the simultaneous analysis of steroids as TMS derivatives.Display Omitted
Keywords: Gas chromatography; Electrospray ionization; Anabolic steroids; Trimethylsilylation;

This paper reports on the advanced development of an ultrasensitive method for the detection of benzene, toluene, and ethylbenzene (or BTE) by low-pressure photoionization mass spectrometry (LPPI-MS). The LPPI source is composed of a laboratory-assembled krypton lamp and a stainless steel cylindrical ionizer. A compact V-shaped mass spectrometer is coupled to the LPPI source with a set of ion immigration optics under dc bias. The fixed standard concentration (FSC) and fixed standard volume (FSV) method are employed to calibrate the sensitivities of the instrument. The corresponding detection sensitivity toward BTE is 4–7 counts/pptv and the 2σ limit of detection (LOD) is 0.5–0.8 part per trillion by volume (pptv). In addition, the measurement accuracy is 95%–105%, and the corresponding precision ranges from 3% to 15% and from 9% to 31% for the FSC and FSV methods, respectively. The stability (standard deviation) of LPPI-MS for a 1 ppbv BTE mixture is less than 0.025 (>12 h). In the detection of BTE, water in ambient air is the most significant interfering factor, leading to the increased background, and inferior LODs of 1–2 pptv for BTE under an RH of ∼90% is observed. Experimental results indicated that LPPI-MS is reliable for the detection of sub-pptv levels of BTE under laboratory conditions.Display Omitted
Keywords: Sub-pptv; LOD; Ultrasensitive; LPPI-MS;

This paper aims at layout optimization design of obstacles in a three-dimensional T-type micromixer. Numerical analysis shows that the direction of flow velocity change constantly due to the obstacles blocking, which produces the chaotic convection and increases species mixing effectively. The orthogonal experiment method was applied for determining the effects of some key parameters on mixing efficiency. The weights in the order are: height of obstacles > geometric shape > symmetry = number of obstacles. Based on the optimized results, a multi-units obstacle micromixer was designed. Compared with T-type micromixer, the multi-units obstacle micromixer is more efficient, and more than 90% mixing efficiency were obtained for a wide range of peclet numbers. It can be demonstrated that the presented optimal design method of obstacles layout in three-dimensional microchannels is a simple and effective technology to improve species mixing in microfluidic devices. The obstacles layout methodology has the potential for applications in chemical engineering and bioengineering.Display Omitted
Keywords: Micromixer; Layout optimization; Chaotic convection; Orthogonal experiment;

Fluorescent nitrogen and sulfur co-doped carbon dots (NSCDs) were synthesized by a one-step pyrolysis strategy using casein as carbon, nitrogen and sulfur sources, and characterized by UV–vis spectrum, fluorescent spectrum, X-ray photoelectron spectroscopy (XPS) and FT-IR, etc. The synthesized NSCDs displayed a blue emission under ultraviolet illumination with a quantum yield of 31.8%, and a good aqueous solubility, photostability and biocompatibility. It was found that the fluorescence intensity of NSCDs could be selectively quenched by Hg2+, so NSCDs was used as an effective probe for the detection of Hg2+. The linear range and the limit of detection (LOD) of the fluorescent sensor based on NSCDs for the detection of Hg2+ were 0.01–0.25 μM and 6.5 nM, respectively. Spiked water samples were detected by the sensor with the recovery of 95.4–106.3% and relative standard deviation (RSD) of 3.6–8.6%. It was also observed that the quenched NSCDs-Hg2+ system could be restored by the addition of biothiols such as l-cysteine (Lcy), homocysteine (Hcy) and glutathione (GSH), thus NSCDs-Hg2+ system was employed as a fluorescent sensor for the detection of biothiols. The linear range and LOD of the NSCDs-Hg2+ system were 1–10 μM and 23.6 nM for Lcy, 0.2–2.5 μM and 12.3 nM for Hcy, and 0.1–2.0 μM and 16.8 nM for GSH, respectively. The NSCDs-Hg2+ system was applied for the detection of biothiols in serum samples with satisfied results. In addition, the study in vitro imaging HeLa cells revealed that the synthesized NSCDs could be used as effective fluorescent probes in cellular imaging without noticeable cytotoxicity.Display Omitted
Keywords: Carbon dots (CDs); Nitrogen and sulfur co-doped carbon dots (NSCDs); Hg2+; Biothiols; Detection; Cellular imaging;

Coexistence of the polymorphic hybrid-1 and hybrid-2 conformers for a given human telomeric G-quadruplex-forming sequence (htG4) complicates its fine structure identification and limits its application as a sensor element. With help from abasic site (AP site)-engineered htG4s serving as the monomorphic representatives of the two typical hybrid conformers, we found that thioflavin T (ThT) can selectively target the hybrid-2 conformer over the hybrid-1 counterpart in monomer and tandem htG4 molecules. The htG4 that solely adopts the monomorphic hybrid-2 conformer engineered by the AP site is most efficient in lighting up ThT fluorescence in K+ and a selective K+ sensor is realized with a remarkable hypersaline-tolerant capability that can work even in 30000-fold excess of Na+. At 600 mM Na+, the dynamic range for K+ detection can be extended to 30 mM with the limit of detection of 20 μM. This is the first report on the fluorescence discrimination of these two hybrid conformers of htG4 although they have long been categorized with their characteristically structural topologies. Our work will attract much interest in the development of sensors based on the monomorphic htG4 conformer since such high performance in sensor development has not been previously achieved.Display Omitted
Keywords: Human telomeric G-quadruplex; Hybrid-1/Hybrid-2; Abasic site; Hypersaline; Fluorescent sensor; Selectivity;

This paper reports a surface functionalization strategy for protein detections based on biotin-derivatized poly(l-lysine)-grafted oligo-ethylene glycol (PLL-g-OEGx-Biotin) copolymers. Such strategy can be used to attach the biomolecule receptors in a reproducible way simply by incubation of the transducer element in a solution containing such copolymers which largely facilitated the sensor functionalization at an industrial scale. As the synthesized copolymers are cationic in physiology pH, surface biotinylation can be easily achieved via electrostatic adsorption on negatively charged sensor surface. Biotinylated receptors can be subsequently attached through well-defined biotin-streptavidin interaction. In this work, the bioactive sensor surfaces were applied for mouse IgG and prostate specific antigen (PSA) detections using quartz crystal microbalance (QCM), optical sensor (BioLayer Interferometry) and conventional ELISA test (colorimetry). A limit of detection (LOD) of 0.5 nM was achieved for PSA detections both in HEPES buffer and serum dilutions in ELISA tests. The synthesized PLL-g-OEGx-Biotin copolymers with different OEG chain length were also compared for their biosensing performance. Moreover, the surface regeneration was achieved by pH stimulation to remove the copolymers and the bonded analytes, while maintaining the sensor reusability as well. Thus, the developed PLL-g-OEGx-Biotin surface assembling strategy is believed to be a versatile surface coating method for protein detections with multi-sensor compatibility.Display Omitted
Keywords: Immuno-sensor; Surface functionalization; Polyelectrolyte; Quartz crystal microbalance; Interferometry; Enzyme-linked immunosorbent assay;

Protocol for the recovery and detection of Escherichia coli in environmental water samples by Ciprian Briciu-Burghina; Brendan Heery; Fiona Regan (178-186).
To achieve active management of bathing areas and to reduce risk associated with the presence of fecal pollution, tests capable of rapid on-site assessment of microbiological water quality are required. A protocol for the recovery and detection of fecal pollution indicator bacteria, E. coli, using β-glucuronidase (GUS) activity was developed. The developed protocol involves two main steps: sample preparation and GUS activity measurement. In the sample preparation step, syringe filters were used with a dual purpose, for the recovery and pre-concentration of E. coli from the water matrix and as μL reactors for bacteria lysis and GUS extraction. Subsequently, GUS activity was measured using a continuous fluorometric method developed previously. The optimum GUS recovery conditions for the sample preparation step were found to be 100 μL PELB (supplemented with 1 mg mL−1 lysozyme and 20 mM DTT) at 37 °C for 30 min. The protocol was evaluated on environmental samples (fresh and seawater) against an establish GUS assay method (Coliplage®). GUS activities corresponding to samples containing as low as 26 MPN E. coli 100 mL−1 were detected for the seawater sample and as low as 110 MPN E. coli 100 mL−1 for the freshwater samples. By comparison with the Coliplage® method, this protocol offered an improvement in the measured GUS activities of 3.1 fold for freshwater samples and 4.1 fold for seawater samples. Furthermore, the protocol developed here, has a time-to-result of 75 min, and successfully addresses the requirement for tests capable of rapid assessment of microbiological water quality.Display Omitted
Keywords: E. coli; β-glucuronidase (GUS); Fecal pollution; Rapid test; Fluorescence;

Novel polymer-based anion-exchangers with covalently-bonded functional layers of quaternized polyethyleneimine for ion chromatography by O.I. Shchukina; A.V. Zatirakha; A.S. Uzhel; A.D. Smolenkov; O.A. Shpigun (187-194).
For the first time novel pellicular anion-exchangers with functional layers of quaternized branched polyethyleneimine (PEI) covalently bonded to the surface of aminated poly(styrene-divinylbenzene) (PS-DVB) with 1,4-butanediol diglycidyl ether (1,4-BDDGE) as a linker are obtained and studied. The proposed method of synthesis includes alkylation of PS-DVB substrate containing secondary aminogroups with 1,4-BDDGE followed by amination with branched PEI containing primary, secondary, and tertiary amino groups. Quaternization of amino groups of PEI required for increasing ion-exchange capacity of the stationary phase is provided with two epoxides – glycidol and 1,4-BDDGE, which results in the considerable variations of ion-exchange selectivity. Chromatographic properties of the obtained anion-exchangers are evaluated using various model mixtures of anions (F, HCOO, CH3COO, C2H5COO, Cl, BrO3 , NO2 , Br, NO3 , ClO3 , S2O3 2−, C2O4 2−, CrO4 2−, SO4 2−, PO4 3−, I, SCN) in hydroxide and carbonate/bicarbonte eluents. All anion-exchangers show better selectivity toward the anions of weakly retained organic acids (acetate, formate, propionate) as compared with previously reported chemically derivatized anion-exchangers with monomeric functional sites in the ion-exchange layer. In terms of separation abilities, novel anion-exchangers with covalently-bonded PEI are not inferior to some commercial columns with grafted polymeric anion-exchange layer. Anion-exchanger quaternized with more hydrophilic glycidol packed in 10-cm long column enables the separation of 16 anions including weakly retained and highly retained polarizable ones within 26 min using gradient elution. Column efficiencies for both anion-exchangers with quaternized PEI are up to 32000 N/m and 37000 N/m in case of hydroxide and carbonate eluent, respectively, which is comparable with the values demonstrated by commercially available columns of related chemistry.Display Omitted
Keywords: Ion chromatography; Anion-exchanger; Branched ion-exchange layer; Cross-linked functional layer; Inorganic anions; Weakly retained organic acids;

Preparation and application of covalently bonded polysaccharide-modified stationary phase for per aqueous liquid chromatography by Tong Chen; Ling Zhu; Huiyuan Lu; Guangsan Song; Yuanyuan Li; Hongbin Zhou; Ping Li; Wanning Zhu; Hechen Xu; Lijun Shao (195-202).
The mixed phosphorylated/methacryloyl polysaccharide derivative was prepared and immobilized onto porous silica surface through the radical polymerization. The successful immobilization of polysaccharide on the silica support was confirmed by FT-IR spectra, elemental analysis and transmission electron microscopy (TEM), and so on. The new stationary phase (MCPP-SP) showed both hydrophilic interaction liquid chromatography (HILIC) and per aqueous liquid chromatography (PALC) characteristics. The chromatographic behaviors were evaluated by investigating the effects of water content, column temperature, mobile phase pH and salt concentration, and a typical PALC retention feature of MCPP-SP based column was observed at high percentage of water content. Compared with C18 column, using MCPP-SP column, separation of polar compounds including synthetic pigments and sulfa compounds in the PALC mode was successfully accomplished. The results demonstrated that MCPP-SP column exhibited stronger retention efficiency for various polar compounds. PALC as a green chromatography analytical method was suitable for the replacement of HILIC.The mixed phosphorylated/methacryloyl polysaccharide derivative was prepared and immobilized onto porous silica surface through the radical polymerization. The new stationary phase (MCPP-SP) showed both hydrophilic interaction liquid chromatography (HILIC) and per aqueous liquid chromatography (PALC) characteristics. Using MCPP-SP column, separation of polar compounds including synthetic pigments and sulfa compounds in the PALC mode was successfully accomplished. The results demonstrated that MCPP-SP column exhibited stronger retention efficiency for various polar compounds.Display Omitted
Keywords: Polysaccharide; Stationary phase; Per aqueous liquid chromatography; Green chromatography;