ALBERT

Description: Stable isotope dilution assays (SIDAs) are becoming ever commoner in mycotoxin analysis, and the number of synthesized or commercially available isotopically labelled compounds has greatly increased in the 7 years since our last review dealing with this topic. Thus, this review is conceived as an update for new applications or improvements of SIDAs for compounds discussed earlier, but the main focus is on newly introduced labelled substances and the development of SIDAs for, for example, fusarin C, moniliformin or the enniatins. Mycotoxin research has concentrated on the emerging group of Alternaria toxins in recent years, and a series of SIDAs have been developed, including ones for tenuazonic acid, alternariol, altertoxins and tentoxin that are discussed in detail in this review. Information about synthetic routes, isotopic purity and mass-spectrometric characterization of labelled compounds is given, as well as about the development and validation of SIDAs and their application to foods, feeds or biological samples. As the number of commercially available labelled standards is increasing continuously, a general tendency for the use of analytical methods based on liquid chromatography coupled with mass spectrometry capable of identifying a series of mycotoxins simultaneously (“multimethods”) and using one or more labelled internal standards can be observed. An overview of these applications is given, thus demonstrating that SIDAs are increasingly being used in routine analysis.

Description: Synchrotron radiation nanoscale computed tomography (SR nano-CT) is a powerful analysis tool and can be used to perform chemical identification, mapping, or speciation of carbon and other elements together with X-ray fluorescence and X-ray absorption near edge structure (XANES) imaging. In practical applications, there are often challenges for SR nano-CT due to the misaligned geometry caused by the sample stage axial vibration. It occurs quite frequently because of experimental constraints from the mechanical error of manufacturing and assembly and the thermal expansion during the time-consuming scanning. The axial vibration will lead to the structure overlap among neighboring layers and degrade imaging results by imposing artifacts into the nano-CT images. It becomes worse for samples with complicated axial structure. In this work, we analyze the influence of axial vibration on nano-CT image by partial derivative. Then, an axial vibration calibration method for SR nano-CT is developed and investigated. It is based on the cross correlation of plane integral curves of the sample at different view angles. This work comprises a numerical study of the method and its experimental verification using a dataset measured with the full-field transmission X-ray microscope nano-CT setup at the beamline 4W1A of the Beijing Synchrotron Radiation Facility. The results demonstrate that the presented method can handle the stage axial vibration. It can work for random axial vibration and needs neither calibration phantom nor additional calibration scanning. It will be helpful for the development and application of synchrotron radiation nano-CT systems.

Description: A selenosugar (selenosugar 1 , methyl-2-acetamido-2-deoxy-1-seleno-β-D-galactopyranoside) was identified in aqueous extracts of muscle tissue of three marine fish species, mackerel ( Scomber scombrus ), sardine ( Sardina pilchardus ), and tuna ( Thunnus albacares ), by high-performance liquid chromatography coupled to elemental and high-resolution molecular mass spectrometry. Selenoneine (2-selenyl-N α , N α , N α -trimethyl-L-histidine), a known selenium compound in fish, was the major form of selenium in the aqueous extracts, and the methylated derivative of selenoneine, namely Se-methylselenoneine, was also identified as a minor natural constituent in the fish. Selenosugar 1 , a major urinary excretion product of selenium often found in organs and body fluids related to selenium excretion, has so far not been reported in muscle tissue. Se-methylselenoneine has been proposed as the main urinary metabolite from selenoneine. This first report of selenosugar 1 and Se-methylselenoneine as natural constituents of fish muscle tissue opens up a new perspective on the role of these compounds in selenium metabolism and is relevant to selenium supplementation studies.

Description: We present a novel strategy based on data-independent acquisition coupled to targeted data extraction for the detection and identification of site-specific modifications of targeted peptides in a completely unbiased manner. This method requires prior knowledge of the site of the modification along the peptide backbone from the protein of interest, but not the mass of the modification. The procedure, named multiplex adduct peptide profiling (MAPP), consists of three steps: 1) A fragment-ion tag is extracted from the data, consisting of the b-type and y-type ion series from the N and C-terminus, respectively, up to the amino-acid position that is believed to be modified; 2) MS1 features are matched to the fragment-ion tag in retention-time space, using the isolation window as a pre-filter to enable calculation of the mass of the modification; and 3) modified fragment ions are overlaid with the unmodified fragment ions to verify the mass calculated in step 2. We discuss the development, applications, and limitations of this new method for detection of unknown peptide modifications. We present an application of the method in profiling adducted peptides derived from abundant proteins in biological fluids with the ultimate objective of detecting biomarkers of exposure to reactive species.

Description: Circulating cell-free DNA (ccfDNA) has great potential for non-invasive diagnostics, and prediction and monitoring of treatment response, but its amount is usually limited. Therefore, the choice of methods to extract and characterize ccfDNA is crucial. In the current study, we performed the most comprehensive comparison of methods for ccfDNA extraction (11 methods), quantification (3 methods), and estimation of the integrity index (2 methods) from small quantities of different kinds of plasma. The QIAamp® Circulating Nucleic Acid Kit and the Norgen Plasma/Serum Circulating DNA Purification Mini Kit showed the best accuracy and reproducibility, but the Norgen kit allowed to extract a higher amount of ccfDNA. This workflow provides a reliable protocol for the multiple applications of ccfDNA in biomedicine. Graphical Abstract Workflow for the evaluation of methods to isolate, quantify and characterize circulating cell-free DNA from small volumes of plasma

Description: After internal contamination, uranium rapidly distributes in the body; up to 20 % of the initial dose is retained in the skeleton, where it remains for years. Several studies suggest that uranium has a deleterious effect on the bone cell system, but little is known regarding the mechanisms leading to accumulation of uranium in bone tissue. We have performed synchrotron radiation-based micro-X-ray fluorescence (SR μ-XRF) studies to assess the initial distribution of uranium within cortical and trabecular bones in contaminated rats’ femurs at the micrometer scale. This sensitive technique with high spatial resolution is the only method available that can be successfully applied, given the small amount of uranium in bone tissue. Uranium was found preferentially located in calcifying zones in exposed rats and rapidly accumulates in the endosteal and periosteal area of femoral metaphyses, in calcifying cartilage and in recently formed bone tissue along trabecular bone. Furthermore, specific localized areas with high accumulation of uranium were observed in regions identified as micro-vessels and on bone trabeculae. These observations are of high importance in the study of the accumulation of uranium in bone tissue, as the generally proposed passive chemical sorption on the surface of the inorganic part (apatite) of bone tissue cannot account for these results. Our study opens original perspectives in the field of exogenous metal bio-mineralization.

Description: The spectroscopic approaches of FTIR imaging and Raman mapping were applied to the characterisation of a new carbon hydroxyapatite/β-glucan composite developed for bone tissue engineering. The composite is an artificial bone material with an apatite-forming ability for the bone repair process. Rabbit bone samples were tested with an implanted bioactive material for a period of several months. Using spectroscopic and chemometric methods, we were able to determine the presence of amides and phosphates and the distribution of lipid-rich domains in the bone tissue, providing an assessment of the composite’s bioactivity. Samples were also imaged in transmission using an infrared microscope combined with a focal plane array detector. CaF 2 lenses were also used on the infrared microscope to improve spectral quality by reducing scattering artefacts, improving chemometric analysis. The presence of collagen and lipids at the bone/composite interface confirmed biocompatibility and demonstrate the suitability of FTIR microscopic imaging with lenses in studying these samples. It confirmed that the composite is a very good background for collagen growth and increases collagen maturity with the time of the bone growth process. The results indicate the bioactive and biocompatible properties of this composite and demonstrate how Raman and FTIR spectroscopic imaging have been used as an effective tool for tissue characterisation.

Description: Cervical cancer is the fourth most common cancer in women worldwide and mainly affects younger women. The mortality associated with cervical cancer can be reduced if the disease is detected at the pre-cancer stage. Current best-practice methods include cytopathology, HPV testing, and histopathology, but these methods are limited in terms of subjectivity, cost, and time. There is an unmet clinical need for new methods to aid clinicians in the early detection of cervical pre-cancer. These methods should be objective and rapid and require minimal sample preparation. Raman spectroscopy is a vibrational spectroscopic technique by which incident radiation is used to induce vibrations in the molecules of a sample and the scattered radiation may be used to characterise the sample in a rapid and non-destructive manner. Raman spectroscopy is sensitive to subtle biochemical changes occurring at the molecular level, enabling spectral variations corresponding to disease onset to be detected. Over the past 15 years, there have been numerous reports revealing the potential of Raman spectroscopy together with multivariate statistical analysis for the detection of a variety of cancers. This paper discusses the recent advances and challenges for cervical-cancer screening and diagnosis and offers some perspectives for the future.

Description: Large arrays of femtoliter-sized chambers are important tools for single molecule research as well as bioanalytical applications. We have optimized the design and fabrication of two array types consisting of 250 × 250 (62 500) femtoliter chambers either by surface etching of fused silica slides or by polydimethylsiloxane (PDMS) molding. Highly diluted solutions of β-galactosidase were enclosed in such arrays to monitor the fluorogenic reactions of hundreds of individual enzyme molecules in parallel by wide-field fluorescence microscopy. An efficient mechanical sealing procedure was developed to prevent diffusion of the fluorescent reaction product out of the chambers. Different approaches for minimizing non-specific surface adsorption were explored. The signal acquisition was optimized to grant both a large field of view and an efficient signal acquisition from each femtoliter chamber. The optimized femtoliter array has enabled a three-in-one enzyme assay system: First, the concentration of active enzyme can be determined in a digital way by counting fluorescent chambers in the array. Second, the activity of the enzyme bulk solution is given by averaging many individual substrate turnover rates without the need for knowing the exact enzyme concentration. Third—unlike conventional enzyme assays—the distribution of individual substrate turnover rates yields insight into the conformational heterogeneity in an enzyme population. The substrate turnover rates of single β-galactosidase molecules were found to be broadly distributed and independent of the type of femtoliter array. In general, both types of femtoliter arrays are highly sensitive platforms for enzyme analysis at the single molecule level and yield consistent results. Graphical Abstract Isolation and analysis of individual enzyme molecules in large arrays of femtoliter-sized chambers

Description: We present a comprehensive discussion of the role that microchip electrophoresis (ME) instrumentation could play in future NASA missions of exploration, as well as the current barriers that must be overcome to make this type of chemical investigation possible. We describe how ME would be able to fill fundamental gaps in our knowledge of the potential for past, present, or future life beyond Earth. Despite the great promise of ME for ultrasensitive portable chemical analysis, to date, it has never been used on a robotic mission of exploration to another world. We provide a current snapshot of the technology readiness level (TRL) of ME instrumentation, where the TRL is the NASA systems engineering metric used to evaluate the maturity of technology, and its fitness for implementation on missions. We explain how the NASA flight implementation process would apply specifically to ME instrumentation, and outline the scientific and technology development issues that must be addressed for ME analyses to be performed successfully on another world. We also outline research demonstrations that could be accomplished by independent researchers to help advance the TRL of ME instrumentation for future exploration missions. The overall approach described here for system development could be readily applied to a wide range of other instrumentation development efforts having broad societal and commercial impact.

Description: Vanadium speciation in the fungus Phycomyces blakesleeanus was examined by X-ray absorption near-edge structure (XANES) spectroscopy, enabling assessment of oxidation states and related molecular symmetries of this transition element in the fungus. The exposure of P. blakesleeanus to two physiologically important vanadium species (V 5+ and V 4+ ) resulted in the accumulation of this metal in central compartments of 24 h old mycelia, most probably in vacuoles. Tetrahedral V 5+ , octahedral V 4+ , and proposed intracellular complexes of V 5+ were detected simultaneously after addition of a physiologically relevant concentration of V 5+ to the mycelium. A substantial fraction of the externally added V 4+ remained mostly in its original form. However, observable variations in the pre-edge-peak intensities in the XANES spectra indicated intracellular complexation and corresponding changes in the molecular coordination symmetry. Vanadate complexation was confirmed by 51 V NMR and Raman spectroscopy, and potential binding compounds including cell-wall constituents (chitosan and/or chitin), (poly)phosphates, DNA, and proteins are proposed. The evidenced vanadate complexation and reduction could also explain the resistance of P. blakesleeanus to high extracellular concentrations of vanadium.

Description: Proton transfer reaction mass spectrometry (PTR-MS) is a well-established technique for real-time analysis of volatile organic compounds (VOCs). Although it is extremely sensitive (with sensitivities of up to 4500 cps/ppbv, limits of detection 〈1 pptv and the response times of approximately 100 ms), the selectivity of PTR-MS is still somewhat limited, as isomers cannot be separated. Recently, selectivity-enhancing measures, such as manipulation of drift tube parameters (reduced electric field strength) and using primary ions other than H 3 O + , such as NO + and O 2 + , have been introduced. However, monoterpenes, which belong to the most important plant VOCs, still cannot be distinguished so more traditional technologies, such as gas chromatography mass spectrometry (GC-MS), have to be utilised. GC-MS is very time consuming (up to 1 h) and cannot be used for real-time analysis. Here, we introduce a sensitive, near-to-real-time method for plant monoterpene research—PTR-MS coupled with fastGC. We successfully separated and identified six of the most abundant monoterpenes in plant studies (α- and β-pinenes, limonene, 3-carene, camphene and myrcene) in less than 80 s, using both standards and conifer branch enclosures (Norway spruce, Scots pine and black pine). Five monoterpenes usually present in Norway spruce samples with a high abundance were separated even when the compound concentrations were diluted to 20 ppbv. Thus, fastGC-PTR-ToF-MS was shown to be an adequate one-instrument solution for plant monoterpene research.

Description: S -Nitrosoglutathione (GSNO) is a very important biomolecule that has crucial functions in many physiological and physiopathological processes. GSNO acts as NO donor and is a candidate for future medicines. This work describes, for the first time, the separation and the detection of GSNO and its decomposition products using capillary electrophoresis coupled to mass spectrometry (CE–MS). The separation was performed in slightly alkaline medium (pH 8.5) under positive-ionization MS detection. The identification of three byproducts of GSNO was formally performed for the first time: oxidized glutathione (GSSG), glutathione sulfinic acid (GSO 2 H), and glutathione sulfonic acid (GSO 3 H). GSO 2 H and GSO 3 H are known to have important biological activity, including inhibition of the glutathione transferase family of enzymes which are responsible for the elimination of many mutagenic, carcinogenic, and pharmacologically active molecules. We observed, after the ageing of GSNO in the solid state, that the proportion of both GSSG and GSO 3 H increases whereas that of GSO 2 H decreases. These results enabled us to propose an oxidation scheme explaining the formation of such products.

Description: A novel method was developed for d / l -isomeric separation of aldopentoses and aldohexoses as their ( S )-(+)-4-( N , N -dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole derivatives using phenylboronate buffer containing sodium dodecyl sulfate as a background electrolyte. The combination of derivatization with a chiral labeling reagent and micellar electrokinetic chromatography with phenylboronate made possible the efficient separation of d / l isomers as well as epimeric isomers of aldopentoses and aldohexoses. Laser-induced fluorescence detection permitted the micromolar-level determination of monosaccharide derivatives. The limit of detection was 105 amol (300 nM), and the repeatabilities of the migration times and peak area responses were 0.8 % and 7.9 % (relative standard deviation; n  = 6), respectively. The method was applied to the determination of d / l - galactose in red seaweed.

Description: The chemical additives used in fracturing fluids can be used as tracers of water contamination caused by hydraulic fracturing operations. For this purpose, a complete chemical characterization is necessary using advanced analytical techniques. Liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC/Q-TOF-MS) was used to identify chemical additives present in flowback and produced waters. Accurate mass measurements of main ions and fragments were used to characterize the major components of fracking fluids. Sodium adducts turned out to be the main molecular adduct ions detected for some additives due to oxygen-rich structures. Among the classes of chemical components analyzed by mass spectrometry include gels (guar gum), biocides (glutaraldehyde and alkyl dimethyl benzyl ammonium chloride), and surfactants (cocamidopropyl dimethylamines, cocamidopropyl hydroxysultaines, and cocamidopropyl derivatives). The capabilities of accurate mass and MS-MS fragmentation are explored for the unequivocal identification of these compounds. A special emphasis is given to the mass spectrometry elucidation approaches used to identify a major class of hydraulic fracturing compounds, surfactants.

Description: Enzyme immunoassays are currently the methods of choice for gluten control in foods labelled as gluten free , providing a mechanism for assessing food safety for consumption by coeliac and other allergic patients. However, their limitations, many of them associated to the reactivity of the different antibodies used and their degree of specificity, have prevented the establishment of a standardised method of analysis. We explore new methods for quantitatively determining gluten content in foods based on the use of two recently described aptamers, raised against a 33-mer peptide recognised as the immunodominant fragment from α2-gliadin. The assays use the target peptide immobilised onto streptavidin-coated magnetic beads in combination with a limited amount of biotin-aptamer in a competitive format, followed by streptavidin-peroxidase labelling of the aptamer that remains bound to the magnetic beads. The enzyme activity onto the beads, measured by chronoamperometry in disposable screen-printed electrodes, is inversely related to the target concentration in the test solution. We find that while the assay using the aptamer with the highest affinity towards the target (Gli 4) achieves low detection limits (~0.5 ppm) and excellent analytical performance, when challenged in samples containing the intact protein, gliadin, it fails in detecting the peptide in solution. This problem is circumvented by employing another aptamer (Gli 1), the most abundant one in the SELEX pool, as a receptor. The proposed assays allow the convenient detection of the allergen in different kinds of food samples, including heat-treated and hydrolysed ones. The obtained results correlate with those of commercially available antibody-based assays, providing an alternative for ensuring the safety and quality of nominally gluten-free foods. Graphical Abstract Electrochemical magnetoassay for gluten determination using biotin-aptamers as receptors

Description: Application of biochar-based fertilizers is increasingly being considered for its potential agronomic and environmental benefits. However, biochar may contain residues of polycyclic aromatic hydrocarbons (PAHs) as a result of its production by pyrolysis. The strong adsorption of PAHs to biochar makes extraction and analysis of biochar-based fertilizers difficult. This study optimizes the extraction of PAHs in biochar-based fertilizer samples by using an ultrasonic bath for quantification by gas chromatography–mass spectrometry. Among 12 solvents, acetone–cyclohexane (1:1) mixture was selected as the optimum solvent for extraction. Three variables affecting the extraction were studied by Box–Behnken design. The optimum conditions were 57 °C extraction temperature, 81 min extraction time, and two extraction cycles, which were validated by assessing the linearity of analysis, LOD, LOQ, recovery, and levels of PAHs in real biochar-based fertilizer samples. Results revealed that the 16 U.S. EPA PAHs had good linearity, with squared correlation coefficients greater than 0.99. LODs were low, ranging from 2.2 ng g −1 (acenaphthene) to 23.55 ng g −1 (indeno[1,2,3- cd ]perylene), and LOQs varied from 7.51 ng g −1 to 78.49 ng g −1 . The recoveries of 16 individual PAHs from the three biochar-based fertilizer samples were 81.8–109.4 %. Graphical Abstract Use of RSM to optimize UAE for extraction of the PAHs in biochar-based fertilizer

Description: Preparative temperature rising elution fractionation (prepTREF) is the standard technique for the preparative fractionation of polyolefins according to crystallisability. For olefin copolymers such as linear low-density polyethylene (LLDPE), it was believed that the TREF elution temperature correlates directly with the copolymer composition. For copolymers having different bulk comonomer contents, the prepTREF fractions of different samples collected at a given temperature were assumed to have the same chemical composition. It was acknowledged quite early that co-crystallisation effects may disturb TREF fractionation and fractions are obtained that are not completely homogeneous. This, however, has not been investigated quantitatively so far. The fundamental statement of prepTREF is challenged for the first time quantitatively using advanced analytical techniques including high-temperature high-performance liquid chromatography (HPLC). Ethylene-1-octene copolymers having bulk comonomer contents ranging from 0.3 to 6.4 mol% were fractionated by prepTREF, and the fractions were analysed by high-temperature size exclusion chromatography, crystallisation analysis fractionation, differential scanning calorimetry and high-temperature HPLC. All analytical results prove that the TREF fractions collected from different samples at the same elution temperature have different chemical compositions. The chemical compositions of the fractions correlate with the compositions of the bulk samples in that the comonomer contents of similar TREF fractions increase with an increase of the comonomer content of the bulk samples. These results are in clear contrast to the previous assumption that the TREF fraction composition is independent of the bulk copolymer composition for a given copolymer type. Graphical abstract Advanced analysis of LLDPE by combination of HT-HPLC, DSC and CRYSTAF

Description: The exploration of new chemical entities from herbal medicines may provide candidates for the in silico screening of drug leads. However, this significant work is hindered by the presence of multiple classes of plant metabolites and many re-discovered structures. This study presents an integrated strategy that uses ultrahigh-performance liquid chromatography/linear ion-trap quadrupole/Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap-MS) coupled with in-house library data for the systematic characterization and discovery of new potentially bioactive molecules. Exploration of the indole alkaloids from Uncaria rhynchophylla (UR) is presented as a model study. Initially, the primary characterization of alkaloids was achieved using mass defect filtering and neutral loss filtering. Subsequently, phytochemical isolation obtained 14 alkaloid compounds as reference standards, including a new one identified as 16,17-dihydro- O -demethylhirsuteine by NMR analyses. The direct-infusion fragmentation behaviors of these isolated alkaloids were studied to provide diagnostic structural information facilitating the rapid differentiation and characterization of four different alkaloid subtypes. Ultimately, after combining the experimental results with a survey of an in-house library containing 129 alkaloids isolated from the Uncaria genus, a total of 92 alkaloids (60 free alkaloids and 32 alkaloid O -glycosides) were identified or tentatively characterized, 56 of which are potential new alkaloids for the Uncaria genus. Hydroxylation on ring A, broad variations in the C-15 side chain, new N -oxides, and numerous O -glycosides, represent the novel features of the newly discovered indole alkaloid structures. These results greatly expand our knowledge of UR chemistry and are useful for the computational screening of potentially bioactive molecules from indole alkaloids. Graphical Abstract A four-step integrated strategy for the systematic characterization and efficient discovery of new indole alkaloids from Uncaria rhynchophylla

Description: Phenylethanolamine A (PA) is a β-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC 50 value of the optimized icELISA was 0.44 ng mL −1 . The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL −1 and 0.39 ng g −1 , respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0–107.4 % and 81.8–113.3 %, respectively, with the coefficients of variation in the range 4.1–16.2 % and 1.2–6.3 %, respectively. The mAbs had negligible cross reactivity with 10 other β-agonists. In contrast, the LFA had a cut-off level of 5 ng mL −1 (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography–tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples. Graphical Abstract Determination of phenylethanolamine A in biological samples by immunological methods

Description: Real-time feedback about dissected tissue during the neurosurgical procedure is strongly requested. A novel direct ionization mass spectrometric method for identifying pathological differences in tissues is proposed. The method is based on simultaneous extraction of tissue lipids and electrospray ionization which allows mass spectrometric data to be obtained directly from soft tissues. The advantage of this method is the stable flow of solvent, which leads to stable time-dependent spectra. The tissues included necrotized tissues and tumor tissues in different combinations. Capability for direct analysis of samples of dissected tissues during the neurosurgical procedure is demonstrated. Data validation is conducted by compound identification using precise masses from the MS profile, MS/MS, and isotopic distribution structure analysis. The method can be upgraded and applied for real-time identification of tissues during surgery. This paper describes the technique and its application perspective. For these purposes, other methods were compared with the investigated one and the results were shown to be reproducible. Differences in lipid profiles were observed even in tissues from one patient where distinctions between different samples could be poor. The paper presents a proof of concept for the method to be applied in neurosurgery particularly and in tissue analysis generically. The paper also contains preliminary results proving the possibility of observing differences in mass spectra of different tumors.

Description: Immunoglobulin Y (IgY) is derived from egg yolk and has been identified as a cheap and high-yield immunoreagent. The application of IgY in immunoassays for the detection of chemical contaminants in food samples has rarely been reported. In this work, we describe a rapid and sensitive fluorescence polarization immunoassay (FPIA) for valnemulin (VAL) using IgY which was produced using a previously prepared immunogen. Three fluorophore-labeled VAL tracers were synthesized and the sensitivity of the best tracer (VAL–DTAF) in the optimized FPIA with antibody IgY 100 demonstrated an IC 50 value of 12 ng mL −1 in buffer. After evaluation of several extraction procedures, acidified acetonitrile was selected to extract VAL from swine tissue. The recoveries of VAL in spiked swine tissue at three levels (50, 100, and 200 μg kg −1 ) were higher than 79 % with coefficients of variation (CVs) lower than 12 %. The limit of detection (LOD) of the FPIA in swine tissue was 26 μg kg −1 and was lower than the maximum residue limit (MRL) of VAL set by the European Union. The study showed that IgY could be a good substitute for IgG when developing a high-throughput assay for chemical residues. Graphical Abstract FPIA based on IgY for rapid detection of Valnemulin in swine tissue

Description: Using Raman and infrared spectroscopy, we monitored spectral changes occurring in the blood plasma of patients with Alzheimer’s disease (AD) in relation to healthy controls. The protein secondary structure as reflected by amide I band involves β-sheet enrichment, which may be attributable to Aβ peptide formation and to increasing proportion of the globulins that are β-sheet rich. Likewise, the behavior of the infrared 1200–1000–cm −1 region and the Raman 980–910- and 450–400-cm −1 regions can be explained in terms of the said plasma composition change. Further, the 744-cm −1 Raman band from healthy control plasma shows frequency upshifting in the course of AD, which may be generated by the platelets collected in blood plasma. Linear discrimination analysis and receiver operating characteristic (ROC) analysis have been used to distinguish between patients with AD and age-matched healthy controls with a diagnostic accuracy of about 94 %. Graphical Abstract ROC assessment of sample classifications by Raman spectroscopy ( dashed line ) and linear combination of infrared and Raman spectroscopies ( solid line )

Description: Molecular imprinting is a very powerful synthetic method for preparation of robust materials with pre-designed molecular selectivity. Molecularly imprinted polymers (MIPs) are attractive substitutes for antibodies in many analytical and bioanalytical applications, e.g. for development of biosensors and for drug assays. In addition to selective molecular binding, new functions are being added to MIPs to make the synthetic materials responsive to different environmental conditions, making it possible to modulate the binding and release of different molecular targets and to simplify affinity separation. Introduction of signal-transduction functionality into MIPs also brings in new, more easily operated chemical sensors for detection and quantification of important analytical targets. Graphical Abstract Multi-functional molecularly imprinted polymers

Description: In the last few decades, MALDI-TOF MS has become a useful technique not only in proteomics, but also as a fast and specific tool for whole cell analysis through intact cell mass spectrometry (IC-MS). The present study evaluated IC-MS as a novel tool for the detection of distinct patterns that can be observed after exposure to a certain toxin or concentration by utilizing the eukaryotic fish cell line RTL-W1. Two different viability assays were performed to define the range for IC-MS investigations, each of which employing copper sulfate, acridine, and β-naphthoflavone (BNF) as model compounds for several classes of environmental toxins. The IC-MS of RTL-W1 cells revealed not only specific spectral patterns for the various toxins, but also that the concentration used had an effect on RTL-W1 profiles. After the exposure with copper sulfate and acridine, the spectra of RTL-W1 showed a significant increase of certain peaks in the higher mass range ( m / z 〉7000), which is probably attributed to the apoptosis of RTL-W1. On the contrary, exposure to BNF showed a distinct change of ion abundances only in the lower mass range ( m / z 〈7000). Furthermore, a set of mass peaks could be identified as a specific biomarker for a single toxin treatment, so IC-MS demonstrates a new method for the distinction of toxic effects in fish cells. Due to fast sample preparation and high throughput, IC-MS offers great potential for ecotoxicological studies to investigate cellular effects of different substances and complex environmental samples. Graphical Abstract Use of intact cell MALDI-TOF mass spectrometry (IC-MS) to detect and differentiate toxic effects of environmental toxins in rainbow trout liver cell line RTL-W1

Description: In this contribution, we investigated whether surface-enhanced Raman scattering (SERS) of serum can be a candidate method for detecting “luminal A” breast cancer (BC) at different stages. We selected three groups of participants aged over 50 years: 20 healthy women, 20 women with early localized small BC, and 20 women affected by BC with lymph node involvement. SERS revealed clear spectral differences between these three groups. A predictive model using principal component analysis (PCA) and linear discriminant analysis (LDA) was developed based on spectral data, and its performance was estimated with cross-validation. PCA-LDA of SERS spectra could distinguish healthy from BC subjects (sensitivity, 92 %; specificity, 85 %), as well as subjects with BC at different stages, with a promising diagnostic performance (sensitivity and specificity, ≥80 %; overall accuracy, 84 %). Our data suggest that SERS spectroscopy of serum, combined with multivariate data analysis, represents a minimally invasive, easy to use, and fast approach to discriminate healthy from BC subjects and even to distinguish BC at different clinical stages.

Description: The presented work reports a generic fluorescent aptasensing design employing carboxy-modified fluorescent particles as a signal-generating probe and magnetic particles as a solid separation support. Carboxy-modified fluorescent particles were used to modify the aptamer and act as a signal-generating probe. Magnetic beads were used as an immobilization surface to perform the function of a solid separation support. As a proof of concept, the assay was used to detect ochratoxin A (OTA). Fluorescent detection based on the displacement and competition format was performed, and the obtained results were compared. The competition-based assays were characterized with improved analytical characteristics as compared to those based on the displacement principle. The competitive fluorescent assays showed a high sensitivity where the detection limit and IC 50 were 0.005 and 7.2 nM respectively. The aptasensing platform was finally demonstrated for the detection of OTA in a beer sample. However, this is a generic approach that can be very easily extended to other matrixes to determine OTA. Additionally, the proposed concept of fluorescent particles as a signal-generating probe in combination with magnetic particles can also be integrated to other fluorescent-based affinity assays.

Description: Over the past decades, several in vitro methods have been tested for their ability to predict either human intestinal absorption (HIA) or penetration across the blood–brain barrier (BBB) of drugs. Micellar liquid chromatography (MLC) has been a successful approach for retention time measurements of drugs to establish models together with other molecular descriptors. Thus far, MLC approaches have only made use of commercial surfactants such as sodium dodecyl sulfate (SDS) and polyoxyethylene (23) lauryl ether (Brij35), which are not representative for the phospholipids present in human membranes. Miltefosine, a phosphocholine-based lipid, is presented here as an alternative surfactant for MLC measurements. By using the obtained retention factors and several computed descriptors for a set of 48 compounds, two models were constructed: one for the prediction of HIA and another for the prediction of penetration across the BBB expressed as log BB. All data were correlated to experimental HIA and log BB values, and the performance of the models was evaluated. Log BB prediction performed better than HIA prediction, although HIA prediction was also improved a lot (from 0.5530 to 0.7175) compared to in silico predicted HIA values.

Description: Aldosterone is a mineralocorticoid steroid hormone, the measurement of which in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Primary hyperaldosteronism is a specifically treatable and potentially curable form of hypertension, which typically presents as drug-resistant hypertension and, in up to 37 % of cases, hypokalemia. Accurate measurement of aldosterone concentration is essential for correct diagnosis. The serum concentrations of aldosterone are in the picomolar range and therefore sensitive aldosterone assays are required. With the advancement in instrumentation of LC-MS/MS, the picomolar range of aldosterone can be easily measured by the newer models, but for those with a less sensitive instrument, special technique for sample preparation to enhance assay sensitivity is required. This work described the use of charge-tagging for the picomolar measurement of serum aldosterone in a less sensitive LC-MS/MS instrument. The assay was linear up to 3000 pmol/L with lower limit of quantitation at 80 pmol/L. The mean relative recovery was 96.5 % with a range of 89.3–101.6 % for aqueous calibrators and the mean relative recovery was 94.8 % with a range of 87.5–101.4 % for serum calibrators. Intra-assay CVs range from 8.2 % to 11.3 %, and inter-assay CVs ranged from 8.5 % to 13.5 % at concentration range from 229 to 1720 pmol/L. The LC-MS/MS method compared well (y = 1.04x + 8.97) with the in-use radioimmunoassay method. There was no significant difference found ( p  = 0.7135) between results determined by LC-MS/MS and radioimmunoassay method.

Description: Molecular heterogeneity of cancer is a major obstacle in tumor diagnosis and treatment. To deal with this heterogeneity, a multidisciplinary combination of different analysis techniques is of urgent need because a combination enables the creation of a multimodal image of a tumor. Here, we develop a computational workflow in order to combine matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) imaging and Raman microspectroscopic imaging for tissue based studies. The computational workflow can be used to confirm a spectral histopathology (SHP) based on one technique with another technique. In this contribution, we confirmed a Raman spectroscopic based SHP with MALDI-imaging. Owing to this combination, we could demonstrate, for a larynx carcinoma sample, that tissue types and different metabolic states could be extracted from the Raman spectra. Further investigations with the help of MALDI spectra yield a better characterization of variable epithelial differentiation and a better understanding of ongoing dysplastic alterations.

Description: A fuzzy chromatography mass spectrometric (FCMS) fingerprinting method combined with chemometric analysis has been established for rapid discrimination of whole-grain flour (WF) from refined wheat flour (RF). Bran, germ, endosperm, and WF from three local cultivars or purchased from a grocery store were studied. The state of refinement (whole vs. refined) of wheat flour was differentiated successfully by use of principal-components analysis (PCA) and soft independent modeling of class analogy (SIMCA), despite potential confounding introduced by wheat class (red vs. white; hard vs. soft) or resources (different brands). Twelve discriminatory variables were putatively identified. Among these, dihexoside, trihexoside, apigenin glycosides, and citric acid had the highest peak intensity for germ. Variable line plots indicated phospholipids were more abundant in endosperm. Samples of RF and WF from three cultivars (Hard Red, Hard White, and Soft White) were physically mixed to furnish 20, 40, 60, and 80 % WF of each cultivar. SIMCA was able to discriminate between 100 %, 80 %, 60 %, 40 %, and 20 % WF and 100 % RF. Partial least-squares (PLS) regression was used for prediction of RF-to-WF ratios in the mixed samples. When PLS models were used the relative prediction errors for RF-to-WF ratios were less than 6 %. Graphical Abstract Workflow of targeting discriminatory compounds by use of FCMS and chemometric analysis

Description: Ammonia in gastric juice is considered a potential biomarker for Helicobacter pylori infection and as a factor contributing to gastric mucosal injury. High ammonia concentrations are also found in patients with chronic renal failure, peptic ulcer disease, and chronic gastritis. Rapid and specific methods for ammonia detection are urgently required by the medical community. Here we present a method to detect ammonia directly in gastric juice based on Fourier transform infrared spectroscopy. The ammonia dissolved in biological liquid samples as ammonium ion was released in air as a gas by the shifting of the pH equilibrium of the ammonium/ammonia reaction and was detected in line by a Fourier transform infrared spectroscopy system equipped with a gas cell for the quantification. The method developed provided high sensitivity and selectivity in ammonia detection both in pure standard solutions and in a simulated gastric juice matrix over the range of diagnostic concentrations tested. Preliminary analyses were also performed on real gastric juice samples from patients with gastric mucosal injury and with symptoms of H. pylori infection, and the results were in agreement with the clinicopathology information. The whole analysis, performed in less than 10 min, can be directly applied on the sample without extraction procedures and it ensures high specificity of detection because of the ammonia fingerprint absorption bands in the infrared spectrum. This method could be easily used with endoscopy instrumentation to provide information in real time and would enable the endoscopist to improve and integrate gastroscopic examinations.

Description: Analytical methods allowing sensitive determination of reduced homocysteine (rHcy), one of the so-called biothiols, in human serum is a topic of growing interest due to its close relation to several human disorders, mainly cardiovascular diseases. Although most widely used analytical strategies to determine total Hcy involve derivatization by means of fluorescent labels, this work proposes the use of ebselen, a Se-containing labelling agent to derivatize the reactive sulfhydryl group of the Hcy molecule in its “free” reduced form, which is more likely to play different roles in disease pathogenesis. Optimization of the derivatization and separation conditions by high-performance liquid chromatography (HPLC) to isolate the excess of derivatizing reagent is carried out here using UV/VIS detection. Further, the study of the Se labelling reaction by electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides a stoichiometry of the derivative of 1:1. The main advantage of using ebselen as a labelling agent is the presence of the Se atom in the molecule that allows the use of inductively coupled plasma mass spectrometry (ICP-MS) as a sensitive and selective Se detector. The coupling of HPLC with ICP-MS provided excellent features for the determination of Se-derivatized rHcy (detection limit of 9.6 nM) in real samples. Quantification was accomplished by using post-column isotope dilution (ID) of Se in serum samples, after precipitation of the main serum proteins. Quantitative results for “free” rHcy turned out to be around 0.18–0.22 μM in serum samples from healthy individuals that could be directly analyzed without sample preconcentration.

Description: A click reaction is reported here for the first time as a useful technique to control the conformational stability of natural organic matter (NOM) suprastructures. Click conjugates were successfully formed between a previously butynylated NOM hydrophobic fraction and a hydrophilic polyethylene glycol (PEG)-amino chain. The click products were shown by size exclusion chromatography (HPSEC) hyphenated with Orbitrap mass spectrometry (MS) in electrospray ionization (ESI) (+), while precursors were visible in ESI (−). Despite their increase in molecular weight, HPSEC elution of click conjugates occurred after that of precursors, thus showing their departure from the NOM supramolecular association. This indicates that the click-conjugated NOM molecules were varied in their hydrophilic and cationic character and lost the capacity to accommodate in the original hydrophobic suprastructures. The most abundant product had the C 16 H 30 O 5 N 4 formula, a click conjugate of butanoic acid, while other products were short-chained (C 4 –C 8 ) linear unsaturated and hydroxylated carboxylic acids. Tandem MS revealed formation of triazole rings in clicked conjugates and their two fragmentations at the ester and the C-N alkyl–aryl bonds. The behavior of NOM molecules modified by click chemistry confirms that hydrophobicity and ionic charge of humic molecules play a pivotal role in stabilizing intermolecular forces in NOM. Moreover, the versatility of the click reaction may become useful to decorate NOM molecules with a variety of substrates, in order to alter NOM conformational and chemical properties and diversify its applications in the environment.

Description: Diacetoxyscirpenol (DAS), a Fusarium mycotoxin belonging to the trichothecene type A mycotoxins, is able to contaminate food and feed worldwide. Only limited information is available regarding the metabolism of DAS. The present study used ultrahigh-performance liquid chromatography-quadrupole/time-of-flight hybrid mass spectrometry (UHPLC-Q/TOF) to investigate the in vitro phase I and II metabolism of DAS by rat, chicken, swine, goat, cow, and human liver microsomes. An extensive metabolization profile of DAS has been observed. A total of seven phase I and three phase II metabolites of DAS were detected. Among the identified molecules, four phase I metabolites (8β-hydroxy-DAS, neosolaniol, 7-hydroxy-DAS, and its epimer) and two phase II metabolites (4-deacetyl-DAS-3-glucuronic acid and 4-deacetyl-DAS-4-glucuronic acid) were identified for the first time. These results indicate that the major metabolic pathways of DAS in vitro were hydrolyzation (M1–M3), hydroxylation (M4–M7), and conjugation (M8–M10). Qualitative differences in phase I and II metabolic profiles of DAS between the five animal species and human were observed. 4-Deacetyl-DAS was the primary metabolite from liver microsomes of all species, especially human. The in vivo metabolism of DAS in rats and chickens after oral administration of DAS was also investigated and compared. The major metabolites for rats and chickens were 4-deacetyl-DAS and 7-hydroxy-DAS. These results will help to gain a more detailed insight into the metabolism and toxicity of DAS among different animal species and human. Graphical Abstract The metabolism of diacetoxyscirpenol in farm animals and human

Description: Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of “click chemistry” reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N -azidoacetylmannosamine (Ac 4 ManNAz) to form N -azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human α-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble β-galactosamide α-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 μM of Ac 4 ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77–82, 2009 ) in the presence of increasing concentrations of Ac 4 ManNAz.

Description: We assessed the clinical feasibility of conducting immunoassays based on surface-enhanced Raman scattering (SERS) in the early diagnosis of rheumatoid arthritis (RA). An autoantibody against citrullinated peptide (anti-CCP) was used as a biomarker, magnetic beads conjugated with CCP were used as substrates, and the SERS nanotags were comprised of anti-human IgG-conjugated hollow gold nanospheres (HGNs). We were able to determine the anti-CCP serum levels successfully by observing the distinctive Raman intensities corresponding to the SERS nanotags. At high concentrations of anti-CCP (〉25 U/mL), the results obtained from the SERS assay confirmed those obtained via an ELISA-based assay. Nevertheless, quantitation via our SERS-based assay is significantly more accurate at low concentrations (〈25 U/mL). In this study, we compared the results of an anti-CCP assay of 74 clinical blood samples obtained from the SERS-based assay to that of a commercial ELISA kit. The results of the anti-CCP-positive group ( n  = 31, 〉25 U/mL) revealed a good correlation between the ELISA and SERS-based assays. However, in the anti-CCP-negative group ( n  = 43, 〈25 U/mL), the SERS-based assay was shown to be more reproducible. Accordingly, we suggest that SERS-based assays are novel and potentially useful tools in the early diagnosis of RA.

Description: We report a comprehensive strategy based on implementation of orthogonal measurement techniques to provide critical and verifiable material characteristics for functionalized gold nanoparticles (AuNPs) used in biomedical applications. Samples were analyzed before and after ≈50 months of cold storage (≈4 °C). Biomedical applications require long-term storage at cold temperatures, which could have an impact on AuNP therapeutics. Thiolated polyethylene glycol (SH-PEG)-conjugated AuNPs with different terminal groups (methyl-, carboxylic-, and amine-) were chosen as a model system due to their high relevancy in biomedical applications. Electrospray-differential mobility analysis, asymmetric-flow field flow fractionation, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, inductively coupled plasma mass spectrometry, and small-angle X-ray scattering were employed to provide both complementary and orthogonal information on (1) particle size and size distribution, (2) particle concentrations, (3) molecular conjugation properties (i.e., conformation and surface packing density), and (4) colloidal stability. Results show that SH-PEGs were conjugated on the surface of AuNPs to form a brush-like polymer corona. The surface packing density of SH-PEG was ≈0.42 nm −2 for the methyl-PEG-SH AuNPs, ≈0.26 nm −2 for the amine-SH-PEG AuNPs, and ≈0.18 nm −2 for the carboxylic-PEG-SH AuNPs before cold storage, approximately 10 % of its theoretical maximum value. The conformation of surface-bound SH-PEGs was then estimated to be in an intermediate state between brush-like and random-coiled, based on the measured thicknesses in liquid and in dry states. By analyzing the change in particle size distribution and number concentration in suspension following cold storage, the long term colloidal stability of AuNPs was shown to be significantly improved via functionalization with SH-PEG, especially in the case of methyl-PEG-SH and carboxylic-PEG-SH (i.e., we estimate that 〉80 % of SH-PEG5K remained on the surface of AuNPs during storage). The work described here provides a generic strategy to track and analyze the material properties of functional AuNPs intended for biomedical applications, and highlights the importance of a multi-technique analysis. The effects of long term storage on the physical state of the particles, and on the stability of the ligand-AuNP conjugates, are employed to demonstrate the capacity of this approach to address critical issues relevant to clinical applications.

Description: An aptamer was previously selected against the anaphylactic allergen β-conglutin (β-CBA I), which was subsequently truncated to an 11-mer and the affinity improved by two orders of magnitude. The work reported here details the selection and characterisation of a second aptamer (β-CBA II) selected against a second aptatope on the β-conglutin target. The affinity of this second aptamer was similar to that of the 11-mer, and its affinity was confirmed by three different techniques at three independent laboratories. This β-CBA II aptamer in combination with the previously selected β-CBA I was then exploited to a dual-aptamer approach. The specific and simultaneous binding of the dual aptamer (β-CBA I and β-CBA II) to different sites of β-conglutin was confirmed using both microscale thermophoresis and surface plasmon resonance where β-CBA II serves as the primary capturing aptamer and β-CBA I or the truncated β-CBA I (11-mer) as the secondary signalling aptamer, which can be further exploited in enzyme-linked aptamer assays and aptasensors.

Description: Previous studies by our group have shown that Sb(V) is able to enter red blood cells in a dynamic process and is reduced to Sb(III) by glutathione. The present study aims to investigate a possible entry pathway for Sb(V) through the erythrocyte membrane. Applying fluorescence spectroscopy studies with Laurdan and diphenylhexatriene (DPH) probes, it was found that there was no interaction between Sb(V) and membrane lipids. By comparing the Sb(V) entry percentages through lipid vesicles and sealed erythrocyte membranes, it was found that Sb(V) required protein channels to pass through the membrane. The competitive inhibition results using HCO 3 − and Cl − showed that the Sb(V) uptake rate through the membrane fell approximately 50–70 % until full inhibition was reached, which was possibly due to the inhibition of the anion exchanger 1 (AE1) channel. Finally, the fluorescence measurements with the 5-iodoacetamidofluorescein (5-IAF) probe showed that Sb(V) interacted with membrane protein SH groups during this process.

Description: Neurotransmitters are an important class of messenger molecules. They govern chemical communication between cells for example in the brain. The spatiotemporal propagation of these chemical signals is a crucial part of communication between cells. Thus, the spatial aspect of neurotransmitter release is equally important as the mere time-resolved measurement of these substances. In conclusion, without tools that provide the necessary spatiotemporal resolution, chemical signaling via neurotransmitters cannot be studied in greater detail. In this review article we provide a critical overview about sensors/probes that are able to monitor neurotransmitters. Our focus are sensing concepts that provide or could in the future provide the spatiotemporal resolution that is necessary to ‘image’ dynamic changes of neurotransmitter concentrations around cells. These requirements set the bar for the type of sensors we discuss. The sensor must be small enough (if possible on the nanoscale) to provide the envisioned spatial resolution and it should allow parallel (spatial) detection. In this article we discuss both optical and electrochemical concepts that meet these criteria. We cover techniques that are based on fluorescent building blocks such as nanomaterials, proteins and organic dyes. Additionally, we review electrochemical array techniques and assess limitations and possible future directions.

Description: Phosphatidylethanol species (PEths) are promising biomarkers of alcohol consumption. Here, we report on the set-up, validation, and application of a novel UHPLC-ESI-MS/MS method for the quantification of PEth 16:0/18:1, PEth 18:1/18:1, and PEth 16:0/16:0 in whole blood (30 μL) and in venous (V, 30 μL) or capillary (C, 3 punches (3 mm)) dried blood spots (DBS). The methods were linear from 10 (LLOQ) to 2000 ng/mL for PEth 16:0/18:1, from 10 (LLOQ) to 1940 ng/mL for PEth 18:1/18:1, and from 19 (LLOQ) to 3872 ng/mL for PEth 16:0/16:0. Extraction efficiencies were higher than 55 % (RSD 〈 18 %) and matrix effects compensated for by IS were between 77 and 125 % (RSD 〈 10 %). Accuracy, repeatability, and intermediate precision fulfilled acceptance criteria (bias and RSD below 13 %). Validity of the procedure for determination of PEth 16:0/18:1 in blood was demonstrated by the successful participation in a proficiency test. The quantification of PEths in C-DBS was not significantly influenced by the hematocrit, punch localization, or spot volume. The stability of PEths in V-DBS stored at room temperature was demonstrated up to 6 months. The method was applied to authentic samples (whole blood, V-DBS, and C-DBS) from 50 inpatients in alcohol withdrawal and 50 control volunteers. Applying a cut-off value to detect inpatients at 221 ng/mL for PEth 16:0/18:1 provided no false positive results and a good sensitivity (86 %). Comparison of quantitative results (Bland-Altman plot, Passing-Bablok regression, and Wilcoxon signed rank test) revealed that V-DBS and C-DBS were valid alternatives to venous blood for the detection of alcohol consumption. Graphical Abstract Passing-Bablok regression analysis of PEth 16:0/18:1 concentrations measured in blood and in capillary DBS, demonstrating equivalence of PEth concentrations in both matrices

Description: A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102–Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

Description: The existence of pharmaceuticals and illicit drugs (PIDs) in environmental waters has led many analytical chemists to develop screening methods for monitoring purposes. Water samples can contain a huge number of possible contaminants, commonly at low concentrations, which makes their detection and identification problematic. Liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) has proven itself effective in the screening of environmental contaminants. The present work investigates the use of the most popular HRMS instruments, quadrupole time-of-flight and linear trap quadrupole-Orbitrap, from two different laboratories. A suspect screening for PIDs was carried out on wastewater (influent and effluent) and surface water samples from Castellón, Eastern Spain, and Cremona, Northern Italy, incorporating a database of 107 PIDs (including 220 fragment ions). A comparison between the findings of both instruments and of the samples was made which highlights the advantages and drawbacks of the strategies applied in each case. In total, 28 compounds were detected and/or identified by either/both instruments with irbesartan, valsartan, benzoylecgonine and caffeine being the most commonly found compounds across all samples. Graphical Abstract A suspect screening of pharmaceuticals and illicit drugs in envrionmental waters of both Castellón, Spain and Cremona, Italy was carried out using liquid chromatography coupled with high resolution mass spectrometry

Description: A fluorescent core-shell molecularly imprinted polymer based on the surface of SiO 2 beads was synthesized and its application in the fluorescence detection of ultra-trace λ -cyhalothrin (LC) was investigated. The shell was prepared by copolymerization of acrylamide with allyl fluorescein in the presence of LC to form recognition sites. The experimental results showed that the thin fluorescent molecularly imprinted polymer (FMIP) film exhibited better selective recognition ability than fluorescent molecularly non-imprinted polymer (FNIP). A new nonlinear relationship between quenching rate and concentration was found in this work. In addition, the nonlinear relationship allowed a lower concentration range of 0–5.0 nM to be described by the Stern–Volmer equation with a correlation coefficient of 0.9929. The experiment results revealed that the SiO 2 @FMIP was satisfactory as a recognition element for determination of LC in soda water samples. Therefore this study demonstrated the potential of MIP for the recognition and detection of LC in food.

Description: Determination of volatile plant compounds in field ambient air is important to understand chemical communication between plants and insects and will aid the development of semiochemicals from plants for pest control. In this study, a thermal desorption–gas chromatography–mass spectrometry (TD–GC–MS) method was developed to measure ultra-trace levels of volatile plant compounds in field ambient air. The desorption parameters of TD, including sorbent tube material, tube desorption temperature, desorption time, and cold trap temperature, were selected and optimized. In GC–MS analysis, the selected ion monitoring mode was used for enhanced sensitivity and selectivity. This method was sufficiently sensitive to detect part-per-trillion levels of volatile plant compounds in field ambient air. Laboratory and field evaluation revealed that the method presented high precision and accuracy. Field studies indicated that the background odor of tea plantations contained some common volatile plant compounds, such as ( Z )-3-hexenol, methyl salicylate, and ( E )-ocimene, at concentrations ranging from 1 to 3400 ng m −3 . In addition, the background odor in summer was more abundant in quality and quantity than in autumn. Relative to previous methods, the TD–GC–MS method is more sensitive, permitting accurate qualitative and quantitative measurements of volatile plant compounds in field ambient air.

Description: Selenium is both essential and toxic for mammals; the range between the two roles is narrow and not only dose-dependent but also related to the chemical species present in foodstuff. Unraveling the metabolism of Se in plants as a function of Se source may thus lead to ways to increase efficiency of fertilization procedures in selenium deficient regions. In this study, stable-isotope tracing was applied for the first time in plants to simultaneously monitor the bio-incorporation of two inorganic Se species commonly used as foodstuff enrichment sources. Occurrence and speciation of Se coming from different Se sources were investigated in root and leaf extracts of ryegrass (Lolium perenne L. ) , which had been co-exposed to two labeled Se species ( 77 SeIV and 82 SeVI). Although the plant absorbed similar amounts of Se when supplied in the form of selenite or selenate, the results evidenced marked differences in speciation and tissues allocation. Selenite was converted into organic forms incorporated mostly into high molecular weight compounds with limited translocation to leaves, whereas selenate was highly mobile being little assimilated into organic forms. Double-spike isotopic tracer methodology makes it possible to compare the metabolism of two species-specific Se sources simultaneously in a single experiment and to analyze Se behavior in not-hyperaccumulator plants, the ICP-MS sensitivity being improved by the use of enriched isotopes.

Description: An analytical method based on micellar liquid chromatography was developed to determine the concentration of three catecholamines (epinephrine, norepinephrine, and dopamine) in urine. The detection of these compounds in urine can be useful to diagnose several diseases, related to stress and sympathoadrenal system dysfunction, using a non-invasive collection procedure. The sample pretreatment was a simple dilution in a micellar solution, filtration, and direct injection, thus avoiding time-consuming and tedious extraction steps. Therefore, there is no need to use an internal standard. The three catecholamines were eluted using a C18 column and a mobile phase of 0.055 M sodium dodecyl sulfate-1.5 % methanol buffered at pH 3.8 running at 1.5 mL/min under isocratic mode in less than 25 min. The detection was performed by amperometry applying a constant potential of +0.5 V. The procedure was validated following the guidelines of the European Medicines Agency in terms of the following: calibration range (0.09–5 μg/mL), linearity ( r 2  〉 0.9995), limit of detection (0.02 μg/mL), within- and between-run accuracy (−6.5 to +8.4 %) and precision (〈10.2 %), dilution integrity, matrix effect, robustness (〈8.4), and stability. The obtained values were below those required by the guide. The method was rapid, easy-to-handle, eco-friendly, and safe and provides reliable quantitative data, and is thus useful for routine analysis. The procedure was applied to the analysis of epinephrine, norepinephrine, and dopamine in urine samples from patients of a local hospital.

Description: A pH probe with a microsecond luminescence lifetime was obtained via covalent coupling of 6-carboxynaphthofluorescein (CNF) moieties to ruthenium-tris-(1,10-phenanthroline) 2+ . The probe was covalently attached to amino-modified poly-(2-hydroxyethyl)methacrylate (pHEMA) and showed a pH-dependent FRET with luminescence lifetimes of 681 to 1260 ns and a working range from ca. pH 6.5 to 9.0 with a pK a of 7.79 ± 0.14. The pH sensor matrix was integrated via spin coating as ca. 1- to 2-μm-thick layer into “CytoCapture” cell culture dishes of 6 mm in diameter. These contained a microcavity array of square-shaped regions of 40 μm length and width and 15 μm depth that was homogeneously coated with the pH sensor matrix. The sensor layer showed fast response times in both directions. A microscopic setup was developed that enabled imaging of the pH inside the microchamber arrays over many hours. As a proof of principle, we monitored the pH of Escherichia coli cell cultures grown in the microchamber arrays. The integrated sensor matrix allowed pH monitoring spatially resolved in every microchamber, and the differences in cell growth between individual chambers could be resolved and quantified. Graphical abstract A pH probe with a microsecond luminescence lifetime is described and its covalent attachment to a hydrogel matrix, integration into microchamber arrays, and use for pH monitoring in a model E. coli miniaturized cell culture.

Description: The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.

Description: Certified reference materials (CRMs) that are compatible with detection methods are needed to detect genetically modified organisms (GMOs). Screening is the first detection step in determining the possible presence of GMO ingredients in food or feed; however, screening has been hindered by the lack of GMO CRMs. In this study, transgenic rice materials were developed via the transformation of a construct harboring 11 commonly used screening elements. Digital PCR was utilized to identify a homozygous single-copy line termed SDrice. The qualitative detections of 11 elements in 21 transgenic materials demonstrated that the genomic DNA of the SDrice was suitable for use as a positive control in the screening of GMO ingredients. The suitability of SDrice as reference material was further checked by testing the sensitivity of 11 known conventional PCR assays, ranging from 10 to 50 copies of the SDrice genome. The standard curves that were created using SDrice DNA series as calibrators all exhibited good linearities in the relationships of the Ct values with the template copy numbers in these 11 real-time PCR assays. The LODs of the real-time PCR assays were estimated to be two to five copies of the SDrice genome. Comparisons of the SDrice with other GM rice revealed that significant differences existed in both the intercepts of the standard curves and the ΔCt values of the exogenous and reference genes for the P-35S, T-nos, HPT , T-35S, and Bar assays; the SDrice was not fit for quantification of other GM rice events. This study provided a matrix reference material (RM) that was suitable for screening GM rice, determination of sensitivity and a LOD of PCR assays, and overcame some of the drawbacks of plasmid DNA as reference material. Graphical Abstract A matrix RM from transgenic rice harboring 11 commonly used screening targets

Description: A rapid, reliable, sensitive, and quantitative multi-residue fluorescent microspheres immunochromatographic assay (FMCA) was developed for simultaneous detection of four macrolides in raw milk. The IC 50 value of the optimized FMCA was 1.36, 1.22, 1.01, and 1.39 ng/mL for erythromycin (ERY), spiramycin (SPI), tilmicosin (TIM), and tylosin (TYL), respectively. The limits of detection (LODs) for the four macrolides was 0.13 ng/mL. The recoveries of ERY, SPI, TIM, and TYL from spiked raw milk ranged from 91.8–109.2, 89.6–114.4, 84.8–111.6, and 85.8–115.2 %, respectively, with coefficients of variation (CVs) of 5.4–11.3, 7.9–15.7, 6.2–13.7, and 3.2–14.9 %, respectively. The whole testing process was completed within 20 min. The antibody-mixed labeled method was successfully applied to the FMCA, which greatly simplified the operation steps and saved a lot of time. Compared with the immunogold chromatographic assay (IGCA), the FMCA is more sensitive and stable and has less antibody consumption. A parallel analysis in blind raw milk samples was conducted by liquid chromatography–tandem mass spectrometry (LC-MS/MS); the results showed good correlation ( r 2  = 0.99) between the two methods. Therefore, the developed multi-residue FMCA is reliable and can be easily applied to other antibiotics or other contaminants. Graphical Abstract Multi-residue FMCA for simultaneous determination of macrolides in raw milk

Description: The water in a solid substrate is generally divided into three forms: hygroscopic, capillary, and free. However, there are few methods available for detecting the contents of different states of water in substrates. In this paper, low-field NMR and MRI were used to analyze the water occurrence characteristics of steam-exploded corn straw in solid-state fermentation (SSF). A significant linear relationship was found between the total NMR peak areas and the total water contents with a correlation coefficient of 0.993. It was further proved to be successful in comparing the contents and distributions of different states of water in static SSF and gas double-dynamic SSF (GDD-SSF). The results showed that among the three states of water, capillary water was the main form of water present and lost in substrates during fermentation. Total water and capillary water contents did not significantly differ as a result of different sample treatments, but hygroscopic water and free water contents in static SSF were respectively 0.38 and 2.98 times that in GDD-SSF with a packing height of 3 cm after fermentation. A relatively uniform water distribution and deep-depth region for microbial growth were found in GDD-SSF, suggesting that GDD-SSF was more suitable for industrialization. This technology has great potential for achieving efficient on-line water supply through water loss detection in SSF. Graphical Abstract Water states and disturibution in SSF measured by low-field NMR and MRI spectroscopy

Description: Many efforts have been made to produce artificial materials with biomimetic properties for applications in binding assays. Among these efforts, the technique of molecular imprinting has received much attention because of the high selectivity obtainable for molecules of interest, robustness of the produced polymers, simple and short synthesis, and excellent cost efficiency. In this review, progress in the field of molecularly imprinted sorbent assays is discussed—with a focus on work conducted from 2005 to date. Graphical Abstract A growing trend in recent years has been the use of molecularly imprinted polymers as replacements for antibodies in various assay formats, as indicated by a steady increase in publications in the area (see graph)

Description: In this work, we present a direct one-step strategy for rapidly preparing dual ligand co-functionalized fluorescent Au nanoclusters (NCs) by using threonine (Thr) and 11-mercaptoundecanoic acid (MUA) as assorted reductants and capping agents in aqueous solution at room temperature. Fluorescence spectra, high-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and infrared (IR) spectroscopy were performed to demonstrate the optical properties and chemical composition of the as-prepared AuNCs. They possess many attractive features such as near-infrared emission (λ em  = 606 nm), a large Stoke's shift (〉300 nm), high colloidal stability (pH, temperature, salt, and time stability), and water dispersibility. Subsequently, the as-prepared AuNCs were used as a versatile probe for “turn off” sensing of Hg 2+ based on aggregation-induced fluorescence quenching and for “turn-on” sensing of oxytetracycline (OTC). This assay provided good linearity ranging from 37.5 to 3750 nM for Hg 2+ and from 0.375 to 12.5 μM for OTC, with detection limits of 8.6 nM and 0.15 μM, respectively. Moreover, the practical application of this assay was further validated by detecting OTC in human serum samples.

Description: With the boom of nanotechnology, nanomaterials (NMs) have been widely utilized in diverse applications, especially in biological and biomedical fields. Understanding how NMs interact with biomolecules, including proteins, DNA, and lipids, is of great importance for revealing the limitations posed and opportunities offered. Model lipid membrane, as a simplified cell membrane model, has been widely used to study the nanomaterial–lipid membrane interactions. In this article, current and emerging techniques, both experimental and theoretical, to investigate the interactions between NMs and model lipid membrane are summarized with each tool’s capacities and limitations, along with future directions and challenges in this exciting area. This critical information will provide methodological guidance for researchers in this field. Graphical Abstract Techniques in NM-model lipid membrane interactions

Description: Blooms of benthic dinoflagellates of the genus Ostreopsis (mainly O. cf. ovata and occasionally O. cf. siamensis ) represent a serious concern for humans in the Mediterranean area, due to production of palytoxin-like compounds listed among the most potent marine toxins known. In this work, six strains of Ostreopsis sp. from Cyprus Island were analyzed through an integrated approach based on molecular, chemical, and eco-toxicological methods. Cypriot Ostreopsis sp. was found to be a species distinct from O. cf. ovata and O. cf. siamensis , belonging to the Atlantic/Mediterranean Ostreopsis spp. clade. Some variability in toxin profiles emerged: three strains produced ovatoxin-a (OVTX-a), OVTX-d, OVTX-e, and isobaric palytoxin, so far found only in O. cf. ovata ; the other three strains produced only new palytoxin-like compounds, which we named ovatoxin-i, ovatoxin-j 1 , ovatoxin-j 2 , and ovatoxin-k. The new ovatoxins present the same carbon skeleton as ovatoxin-a, differing primarily in an additional C 2 H 2 O 2 moiety and an unsaturation in the region C49–C52. Other minor structural differences were found, including the presence of a hydroxyl group at C44 (in OVTX-j 1 and OVTX-k) and the lack of a hydroxyl group in the region C53–C78 (in OVTX-i and OVTX-j 1 ). The toxin content of the analyzed Ostreopsis sp. strains was in the range 0.06–2.8 pg cell −1 , definitely lower than that of a Ligurian O. cf. ovata strain cultured under the same conditions. Accordingly, an eco-toxicological test on Artemia salina nauplii demonstrated that Ostreopsis sp. presents a very low toxicity compared to O. cf. ovata . The whole of these data suggest that Ostreopsis sp. from Cyprus Island poses a relatively low risk to humans.

Description: Botulinum neurotoxins (BoNT) are divided into seven toxinotypes based on their immunological properties and each toxinotype contains several subtypes according to their amino acid sequences. Here, we designed a mass spectrometry method able to identify BoNT/A subtypes in complex matrices including crude culture supernatants, food, and environmental samples. Peptides from BoNT light chain (L) specific to the subtypes BoNT/A1 to A3 and BoNT/A5 to A8 were identified. The method consists of an immunocapture step with antibodies specific to BoNT/A L chains followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) on a triple quadrupole mass spectrometer (QqQ) in multiple reaction monitoring (MRM) mode. BoNT/A subtypes were correctly identified in culture supernatants and in tap water or orange juice samples with a limit of detection of 20 to 150 mouse lethal doses (MLD) and with a lower sensitivity in serum samples.

Description: Evaluation and diagnosis of blood alterations is a common request for clinical laboratories, requiring a complex technological approach and dedication of health resources. In this paper, we present a microfluidic device that owing to a novel combination of hydrodynamic and dielectrophoretic techniques can separate plasma from fresh blood in a microfluidic channel and for the first time allows optical real-time monitoring of the components of plasma without pre- or post-processing. The microchannel is based on a set of dead-end branches at each side and is initially filled using capillary forces with a 2-μL droplet of fresh blood. During this process, stagnation zones are generated at the dead-end branches and some red blood cells (RBCs) are trapped there. An electric field is then applied and dielectrophoretic trapping of RBCs is used to prevent more RBCs entering into the channel, which works like a sieve. Besides, an electroosmotic flow is generated to sweep the rest of the RBCs from the central part of the channel. Consequently, an RBC-free zone of plasma is formed in the middle of the channel, allowing real-time monitoring of the platelet behavior. To study the generation of stagnation zones and to ensure RBC trapping in the initial constrictions, two numerical models were solved. The proposed experimental design separates up to 0.1 μL blood plasma from a 2-μL fresh human blood droplet. In this study, a plasma purity of 99 % was achieved after 7 min, according to the measurements taken by image analysis. Graphical Abstract Schematics of a real-time plasma monitoring system based on a Hydrodynamic and direct-current insulator-based dielectrophoresis microfluidic channel

Description: The interactions between insects and pathogenic fungi are complex. We employed metabolomic techniques to profile insect metabolic dynamics upon infection by the pathogenic fungus Beauveria bassiana . Silkworm larvae were infected with fungal spores and microscopic observations demonstrated that the exhaustion of insect hemocytes was coupled with fungal propagation in the insect body cavity. Metabolomic analyses revealed that fungal infection could significantly alter insect energy and nutrient metabolisms as well as the immune defense responses, including the upregulation of carbohydrates, amino acids, fatty acids, and lipids, but the downregulation of eicosanoids and amines. The insect antifeedant effect of the fungal infection was evident with the reduced level of maclurin (a component of mulberry leaves) in infected insects but elevated accumulations in control insects. Insecticidal and cytotoxic mycotoxins like oosporein and beauveriolides were also detected in insects at the later stages of infection. Taken together, the metabolomics data suggest that insect immune responses are energy-cost reactions and the strategies of nutrient deprivation, inhibition of host immune responses, and toxin production would be jointly employed by the fungus to kill insects. The data obtained in this study will facilitate future functional studies of genes and pathways associated with insect–fungus interactions.

Description: Designer piperazines are emerging novel psychoactive substances (NPS) with few high-throughput screening methods for their identification. We evaluated a biochip array technology (BAT) immunoassay for phenylpiperazines (PNP) and benzylpiperazines (BZP) and analyzed 20,017 randomly collected urine workplace specimens. Immunoassay performance at recommended cutoffs was evaluated for PNPI (5 μg/L), PNPII (7.5 μg/L), and BZP (5 μg/L) antibodies. Eight hundred forty positive and 206 randomly selected presumptive negative specimens were confirmed by liquid chromatography high-resolution mass spectrometry (LC-HRMS). Assay limits of detection for PNPI, PNPII, and BZP were 2.9, 6.3, and 2.1 μg/L, respectively. Calibration curves were linear ( R 2  〉 0.99) with upper limits of 42 μg/L for PNPI/PNII and 100 μg/L for BZP. Quality control samples demonstrated imprecision 〈19.3 %CV and accuracies 86.0–94.5 % of target. There were no interferences from 106 non-piperazine substances. Seventy-eight of 840 presumptive positive specimens (9.3 %) were LC-HRMS positive, with 72 positive for 1-(3-chlorophenyl)piperazine (mCPP), a designer piperazine and antidepressant trazodone metabolite. Of 206 presumptive negative specimens, one confirmed positive for mCPP (3.3 μg/L) and one for BZP (3.6 μg/L). BAT specificity (21.1 to 91.4 %) and efficiency (27.0 to 91.6 %) increased, and sensitivity slightly decreased (97.5 to 93.8 %) with optimized cutoffs of 25 μg/L PNPI, 42 μg/L PNPI, and 100 μg/L BZP. A high-throughput screening method is needed to identify piperazine NPS. We evaluated performance of the Randox BAT immunoassay to identify urinary piperazines and documented improved performance when antibody cutoffs were raised. In addition, in randomized workplace urine specimens, all but two positive specimens contained mCPP and/or trazodone, most likely from legitimate medical prescriptions. Graphical Abstract Biochip array technology (BAT) immunoassay for designer piperazines detection in urine. In chemiluminescent immunoassay, the labeled-drug (antigen) competes with the drug in the urine. In the absence of drug, the labeled-drug binds to the antibody releasing an enzyme (horseradish peroxidase) to react with the substrate and producing chemiluminescence. The higher the drug concentration in urine, the weaker the chemiluminescent signal is produced. All presumptive positive specimens and randomly selected presumptive negative specimens were analyzed and confirmed by a liquid chromatography high-resolution mass spectrometry with limit of quantification of 2.5 or 5 μg/L

Description: Most methods reported in the literature for determination of nitrofuran metabolites use the same extraction and derivatisation conditions to hydrolyse and extract the protein-bound residues from animal tissue. While undertaking certification of reference materials for nitrofuran metabolites in freeze-dried prawn, it was found that these conditions are satisfactory for recovery of spiked residues; however, extraction efficiencies of incurred furazolidinone could be substantially increased by further optimisation of the extraction conditions. The availability of a suitable certified reference material allows laboratories to ensure that their method is optimised for incurred residues.

Description: Nitroaromatic compounds, for example trinitrotoluene and 2,4,6-trinitrophenol (TNP), are well-known primary constituents of many unexploded landmines worldwide. These compounds are recognized as environmental contaminants and as toxic to living organisms. Therefore, Förster resonance energy transfer (FRET) for TNP detection was developed on the basis of spectral overlap between the fluorescence spectrum of copper nanoclusters (CuNCs) and absorption spectrum of TNP (the calculated Förster distance R 0 of the donor CuNCs and the acceptor TNP is 2.8 nm). Water-soluble fluorescent CuNCs capped with bovine serum albumin have fluorescence emission from 350 nm to 500 nm with maximum fluorescence emission at 400 nm, which overlaps with the absorption spectra of TNP from 350 nm to 450 nm. Inspired by FRET structures, an unprecedented energy-donor-and-acceptor pair of fluorescent CuNCs and TNP is developed in this work. Fluorescence of CuNCs is quenched in the presence of TNP as a result of FRET from fluorescent CuNCs to TNP. Therefore a fluorescence quenching method for the determination of TNP is developed. It achieves TNP detection from 0.8 μmol L −1 to 100 μmol L −1 , with response within 1 min and with good selectivity compared with that for other nitroaromatic compounds, including 2,4-dinitrotoluene, p -nitrotoluene, and nitrobenzene, and phenol. Graphical Abstract Förster resonance-energy-transfer detection of 2,4,6-trinitrophenol using copper nanoclusters was first developed. It achieves TNP detection from 0.8 mmol L −1 to 100 mmol L −1 within 1 min with good selectivity compared with other nitroaromatic compounds

Description: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the method of choice for quantifying small molecules in research and clinical setting. Although there is a large toolkit to increase quantification levels for LC-MS/MS, these techniques are sometimes insufficient to attain the needed limits of quantification (LOQs) or the method becomes too impractical for routine use. We examined the possibilities and limitations of signal summing, an under-utilized, easy-to-apply practice to increase LOQs for an immunosuppressant LC-MS/MS method. The limits of signal summing for everolimus were tested by running samples of everolimus at three concentrations in triplicate programming, increasing amounts of identical transitions in a constant cycle time up to the maximum number the software permitted to sum. The increase in peak area and the signal-to-noise ratio were determined. The effect on imprecision of peak areas and response ratios was evaluated by injection of a low concentration of everolimus tenfold using respectively one and five identical transitions, retaining an identical ion counting time. We compared the imprecision, LOQ, and recovery for our routine everolimus method (using one transition for everolimus and one for d3-everolimus) and an adapted method summing three identical transitions for everolimus (and one for d3-everolimus). The increase in signal was close to the theoretically expected one with a larger experimental spread for everolimus once more than five transitions were used. There was no clear beneficial effect of summing on imprecision. The adapted everolimus method showed a lower LOQ, but comparable imprecision and recovery as the routine method. Quantification levels can be improved by signal summing. No clear effect on imprecision was observed.

Description: Analytical techniques for detection of ochratoxin A (OTA) in food products and blood serum are of great significance. In this study, a fluorescent aptasensor was developed for sensitive and specific detection of OTA, based on a DNA pyramid nanostructure (DPN) and PicoGreen (PG) dye. The designed aptasensor inherits characteristics of DPN, such as high stability and capacity for PG loading. PG, as a fluorescent dye, could bind to double-stranded DNA (dsDNA). In the absence of OTA, the pyramid structure of DPN remains intact, leading to a very strong fluorescence emission. Because of higher affinity of aptamer for its target relative to its complementary strand, upon addition of target, the pyramid structure of DPN is disassembled, leading to a weak fluorescence emission. The presented aptasensor showed high specificity toward OTA with a limit of detection (LOD) as low as 0.135 nM. Besides, the designed sensing strategy was successfully utilized to recognize OTA in serum and grape juice with LODs of 0.184 and 0.149 nM, respectively.

Description: The nonpathogenic filamentous fungus Scedosporium dehoogii was used for the first time to study the electrochemical biodegradation of acetaminophen (APAP). A carbon fiber microelectrode (CFME) modified by nickel tetrasulfonated phthalocyanine (p-NiTSPc) and a carbon paste electrode (CPE) modified with coffee husks (CH) were prepared to follow the kinetics of APAP biodegradation. The electrochemical response of APAP at both electrodes was studied by cyclic voltammetry and square wave voltammetry. p-NiTSPc-CFME was suitable to measure high concentrations of APAP, whereas CH–CPE gave rise to high current densities but was subject to the passivation phenomenon. p-NiTSPc–CFME was then successfully applied as a sensor to describe the kinetics of APAP biodegradation: this was found to be of first order with a kinetics constant of 0.11 day −1 (at 25 °C) and a half-life of 6.30 days. APAP biodegradation by the fungus did not lead to the formation of p -aminophenol (PAP) and hydroquinone (HQ) that are carcinogenic, mutagenic, and reprotoxic (CMR). Graphical Abstract The kinetics of APAP biodegradation, followed by a poly-nickel tetrasulfonated phtalocyanine modified carbon fiber microelectrode

Description: A nano-silver electrode immobilizing acetylcholinesterase (AChE) for the detection of organophosphorus (OPPs) pesticides is reported. Scanning electron microscopy (SEM) was used to characterize the surface structure of two kinds of electrodes fabricated with different sizes of silver powders and the interface between chitosan layer and nano-silver powder layer. Cyclic voltammetry was carried out to characterize the response of silver/chitosan electrode in the absence and in the presence of thiocholine (TCh). It was also used to evaluate the insulativity of the chitosan layer. An amperometric method was performed to measure the response of the electrode to TCh, which is the product of the enzymatic reaction for detecting organophosphorus pesticides indirectly. Although there are many kinds of nanoparticles, silver was chosen for its internal advantage in detecting TCh at low potential without further modification. The result shows nano-silver powder has better performance than usual silver powder, and the limit of detection of paraoxon is 4 ppb under optimized conditions. One percent (w/v) chitosan solution was used as binder for the immobilization of nano-silver powder and AChE, which made it possible for independent electrode fabrication at room temperature, whereas 3% (w/v) chitosan solution was used as insulating compound for controlling the electrode area. Unlike traditional organic insulating ink, chitosan is safe and environmentally friendly, and it is used as insulating material for the first time. The flexible nano-silver/AChE/chitosan electrode was evaluated in Chinese chives and cabbage , and the recoveries of standard addition were 105.11 and 96.41%, respectively. Owing to the antibacterial property of nano-silver and the biocompatibility, safety, and biodegradability of chitosan, the proposed method is safe, facile, environmentally friendly, and has great potential in organophosphorus pesticide detection for food safety. Graphical Abstract Current response of nano-silver electrode ( a ) and silver electrode ( b ) to thiocholine in 0.02 M PBS + KCl at 0.15 V; addition of thiocholine (0.09 mM) every 50 s (↓); inset: calibration curve of nano-silver (▲) and silver (◆) electrode

Description: A procedure is described that results in a substantial increase in signal intensity and in improved accuracy of positive-ion mass calibration when using commercially available kits of monodisperse dendrimers (SpheriCal ® ) in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The peak intensities are amplified by an admixture of 2-[(2 E )-3-(4- tert -butylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) matrix to the kits comprising of 9-nitroanthracene matrix, sodium trifluoroacetate, and four dendrimers. Boosted ion formation then permits lower laser fluence to be used and thus yields enhanced mass resolution. Further, the number of reference peaks is doubled by doping the sample preparation with cesium ions. This results in four [M+Cs] + ion signals in addition to four [M+Na] + ion signals provided by the standard kit. Overall, the modified procedure notably reduces the consumption of the expensive calibration standard kits, while it increases mass resolution and enables the use of an advanced calibration algorithm requiring at least six reference peaks. Graphical abstract A dendrimer-based mass calibration for MALDI-TOF-MS can be improved by adding a DCTB matrix and doping the sample preparation with Cs + ions. Having eight rather than just four reference peaks reduces the average mass error of the calibration curve about fivefold.

Description: Protein glycosylation plays a key role in many biological processes. In this study, a novel carbon material with nanopores was prepared by carbonization of metal–organic framework (MOF) Mil-101(Cr). The parent MOF assembled from metal ions with bridging organic linkers had many fascinating properties, such as ultrahigh surface area, suitable nanopore structure, and especially a large amount of carbon after being calcined. Due to the strong interactions between carbon and glycans as well as the size-exclusion effect of pore against protein, the N-linked glycans from standard glycoprotein or complex human serum proteins could be identified with high efficiency. The simple synthesis method as well as good enrichment efficiency made this novel carbon material a promising tool for glycosylation research.

Description: MicroRNAs (miRNAs) are currently considered as potential biomarkers for various human diseases. In the present study, miRNA-triggered real-time fluorescent isothermal reaction with exponential amplification (ReFIRE) with or without Thermus aquaticus MutS ( Taq MutS) was developed to analyze miRNAs using DNA polymerase, a nicking endonuclease, and fluorescently labeled primers. In the absence of Taq MutS, the ReFIRE system permitted the detection of 100 ymol of targeted miRNA in 80 min. However, this system enabled limited differentiation between homologous miRNA family members. Upon addition of Taq MutS to the ReFIRE system, non-specific amplification generated from the mishybridization between primers and primer dimers or primers and the template duplex was eliminated. The addition of Taq MutS enabled the ultrasensitive detection of as little as 10 ymol of targeted miRNAs in 50 min, which corresponds to less than 10 copies of miRNAs in a total volume of 20 μl. Additionally, the assay exhibited a dynamic range of up to 12 orders of magnitude. The ReFIRE system also showed high specificity, enabling differentiation between homologous miRNA family members exhibiting only single-base differences. The sensitivity, specificity, and dynamic range associated with this system were greater than most currently available miRNA isothermal amplification assays. Moreover, when target-specific primers were labeled with different fluorescent reporters, multiplex analysis was easily performed in a single tube, permitting accurate normalization of miRNA expression. This simple, fast, ultrasensitive, highly specific, and easy-to-multiplex method could significantly contribute to research investigations pertaining to the biological roles of miRNA, as well as clinical diagnosis of various diseases that involve miRNA disruptions. Graphical Abstract The principle of ReFIRE system

Description: Bioconjugation and functionalization of polymer surfaces are two major tasks in materials chemistry which are accomplished using a variety of coupling agents. Immobilization of biomolecules onto polymer surfaces and the construction of bioconjugates are essential requirements of many biochemical assays and chemical syntheses. Different linkers with a variety of functional groups are used for these purposes. Among them, the benzophenones, aryldiazirines, and arylazides represent the most commonly used photolinker to produce the desired chemical linkage upon their photo-irradiation. In this review, we describe the versatile applications of 4-fluoro-3-nitrophenyl azide, one of the oldest photolinkers used for photoaffinity labeling in the late 1960s. Surprisingly, this photolinker, historically known as 1-fluoro-2-nitro-4-azidobenzene (FNAB), has remained unexplored for a long time because of apprehension that FNAB forms ring-expanded dehydroazepine as a major product and hence cannot activate an inert polymer. The first evidence of photochemical activation of an inert surface by FNAB through nitrene insertion reaction was reported in 2001, and the FNAB-activated surface was found to conjugate a biomolecule without any catalyst, reagent, or modification. FNAB has distinct advantages over perfluorophenyl azide derivatives, which are contemporary nitrene-generating photolinkers, because of its simple, single-step preparation and ease of thermochemical and photochemical reactions with versatile polymers and biomolecules. Covering these aspects, the present review highlights the flexible chemistry of FNAB and its applications in the field of surface engineering, immobilization of biomolecules such as antibodies, enzymes, cells, carbohydrates, oligonucleotides, and DNA aptamers, and rapid diagnostics. Graphical Abstract An overview of the FNAB-engineered activated polymer surfaces for covalent ligation of versatile biomolecules

Description: Bisphenol A (BPA) is an important industrial chemical used as a plasticizer in polycarbonate and epoxy resins in the plastic and paper industries. Because of its estrogenic properties, BPA has attracted increasing attention from many researchers. This review focuses primarily on analytical methods for BPA detection that have emerged in recent years. We present and discuss the advantages and disadvantages of sample preparation techniques (e.g., solvent extraction, solid-phase extraction, molecularly imprinted polymer solid-phase extraction, and micro-extraction techniques) and analytical methods (e.g., liquid chromatography, liquid chromatography−mass spectrometry, gas chromatography−mass spectrometry, capillary electrophoresis, immunoassay, and several novel sensors). We also discuss expected future developments for the detection of BPA. Graphical Abstract This review focuses primarily on the recent development in the detection of bisphenol A including sample pre-treatment and analytical methods

Description: If the biomarker potential of intact heteromers and their free subunits is different, differentiation between these forms may reveal important clinical information. Such differentiation may however be analytically challenging. One possible way of circumventing this challenge is by performing a dual-immuno-MS approach. In the present paper, a two-step immunoaffinity sample preparation step is succeeded by digestion and subsequent LC-MS analysis to provide high-sensitivity quantification and differentiation between the heterodimer human chorionic gonadotropin (hCG) and its free β-subunit in serum. Intact and free variants are captured in two separate immunoextraction steps in order to increase the differentiation power of the method. Intact heterodimer variants were depleted prior to free subunit variants in order to incorporate a method quality control. The method was optimized for serum samples. A fully validated immuno-MS method was used as foundation, and partial validation according to the European Medicines Agency’s (EMA) guidelines on validation of bioanalytical methods was performed for the dual approach. An accelerated digestion step was incorporated making batch processing of samples within 1 day possible (approx. 3.5 h of sample preparation including digestion). Acceptable linearity ( R 2  ≥ 0.990 for four variants and R 2 of 0.920 and 0.966 for the remaining two) and specificity were demonstrated, and the method was robust toward varying levels of intact heterodimer versus free subunit. The method was also successfully tested on realistic samples, demonstrating both the differences in total hCG and the distribution between intact hCG and its free β-subunit in real samples. Graphical abstract Schematic overview of the dual immuno-MS process

Description: The online combination of capillary electrophoresis (CE) with mass spectrometry (MS) has long been desired for the capability of direct and simultaneous separation and detection with high efficiency, accuracy, and throughput. In this work, a novel CE-MS interface was developed, using dielectric barrier discharge ionization (DBDI). The interface employed a spray tip with a coaxial three-layer structure, into which the CE sample solution, the sheath liquid, and the nebulizing gas were introduced. The spray tip was put between the DBDI outlet and the MS inlet, thus the CE sample solution could be blended with the sheath liquid, then nebulized. The nebulized sample could be ionized by DBDI, and finally analyzed by MS. The key parameters of the interface were optimized. Then, proof-of-concept experiments separating and detecting the mixture of metronidazole and acetaminophen solutions were conducted. The results showed high separation efficiency, low time consumption, high reproducibility, and convenience in operation. In addition, the interface exhibited a high tolerance of non-volatile salts and surfactants, which would be widely used in CE analyses. All of these results demonstrated that the newly developed CE-DBDI-MS interface could be successfully used in CE-MS studies, and could be further utilized in multiple areas involving efficient separation and detection.

Description: Adoption of Quality by Design (QbD) principles, regulatory support of QbD, process analytical technology (PAT), and continuous manufacturing are major factors effecting new approaches to pharmaceutical manufacturing and bioprocessing. In this review, we highlight new technology developments, data analysis models, and applications of Raman spectroscopy, which have expanded the scope of Raman spectroscopy as a process analytical technology. Emerging technologies such as transmission and enhanced reflection Raman, and new approaches to using available technologies, expand the scope of Raman spectroscopy in pharmaceutical manufacturing, and now Raman spectroscopy is successfully integrated into real-time release testing, continuous manufacturing, and statistical process control. Since the last major review of Raman as a pharmaceutical PAT in 2010, many new Raman applications in bioprocessing have emerged. Exciting reports of in situ Raman spectroscopy in bioprocesses complement a growing scientific field of biological and biomedical Raman spectroscopy. Raman spectroscopy has made a positive impact as a process analytical and control tool for pharmaceutical manufacturing and bioprocessing, with demonstrated scientific and financial benefits throughout a product’s lifecycle.

Description: Our greater understanding of the importance of N -linked glycosylation in biological systems has spawned the field of glycomics and development of analytical tools to address the many challenges regarding our ability to characterize and quantify this complex and important modification as it relates to biological function. One of the unmet needs of the field remains a systematic method for characterization of glycans in new biological systems. This study presents a novel workflow for identification of glycans using Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT™) strategy developed in our lab. This consists of monoisotopic mass extraction followed by peak pair identification of tagged glycans from a theoretical library using an in-house program. Identification and relative quantification could then be performed using the freely available bioinformatics tool Skyline. These studies were performed in the biological context of studying the N- linked glycome of differentiating xylem of the poplar tree, a widely studied model woody plant, particularly with respect to understanding lignin biosynthesis during wood formation. Through our workflow, we were able to identify 502 glycosylated proteins including 12 monolignol enzymes and 1 peroxidase (PO) through deamidation glycosite analysis. Finally, our novel semi-automated workflow allowed for rapid identification of 27 glycans by intact mass and by NAT/SIL peak pairing from a library containing 1573 potential glycans, eliminating the need for extensive manual analysis. Implementing Skyline for relative glycan quantification allowed for improved accuracy and precision of quantitative measurements over current processing tools which we attribute to superior algorithms correction for baseline variation and MS1 peak filtering. Graphical abstract Workflow for FANGS-INLIGHT glycosite profiling of plant xylem and monolignol proteins followed by INLIGHT tagging with semi-automated identification of glycans by light-heavy peak pairs. Finally, manual validation and relative quantification was performed in Skyline

Description: A novel diffusive gradients in thin films (DGT) technique for sampling labile soil sulfate was developed, based on a strong basic anion exchange resin (Amberlite IRA-400) for sulfate immobilization on the binding gel. For reducing the sulfate background on the resin gels, photopolymerization was applied instead of ammonium persulfate-induced polymerization. Agarose cross-linked polyacrylamide (APA) hydrogels were used as diffusive layer. The sulfate diffusion coefficient in APA gel was determined as 9.83 × 10 −6  ± 0.35 × 10 -6 cm 2  s −1 at 25 °C. The accumulated sulfate was eluted in 1 mol L −1 HNO 3 with a recovery of 90.9 ± 1.6 %. The developed method was tested against two standard extraction methods for soil sulfate measurement. The obtained low correlation coefficients indicate that DGT and conventional soil test methods assess differential soil sulfate pools, rendering DGT a potentially important tool for measuring labile soil sulfate.

Description: Selenium nanoparticles (SeNPs) were incorporated in a flexible multilayer plastic material using a water-base adhesive as vehicle for SeNPs. The antioxidant performance of the original solutions containing spherical SeNPs of 50–60 nm diameter, the adhesive containing these SeNPs, and the final multilayer plastic material to be used as food packaging were quantitatively measured. The radical scavenging capacity due to SeNPs was quantified by a free radical assay developed in the laboratory and by the diphenyl-1-picrylhydrazyl (DPPH) method. DPPH was not efficient to measure the scavenging capacity in the multilayer when the free radical scavenger is not in the surface in contact with it. Several multilayer laminated structures composed by [PET (20 m)–adhesive–LDPE (with variable thickness from 35 to 90 μm)] were prepared and measured, demonstrating for the first time that free radicals derived from oxygen (OH·, O 2 ·, and O 2 H) cross the PE layer and arrive at the adhesive. SeNPs remain as such after manufacture and the final laminate is stable after 3 months of storage. The antioxidant multilayer is a non-migrating efficient free radical scavenger, able to protect the packaged product versus oxidation and extending the shelf life without being in direct contact with the product. Migration tests of both Se and SeNPs to simulants and hazelnuts demonstrated the non-migrating performance of this new active packaging. Graphical abstract ᅟ

Description: A new sample extraction procedure based on micro-solid-phase extraction (μSPE) using a mixture of sorbents of different polarities (polymeric reversed-phase sorbent HLB, silica-based sorbent C 18 , and multiwalled carbon nanotubes) was applied to extract benzene, toluene, butyraldehyde, benzaldehyde, and tolualdehyde present in saliva to avoid interference from moisture and matrix components and enhance sensitivity and selectivity of the ion mobility spectrometry (IMS) methodology proposed. The extraction of target analytes from saliva samples by using μSPE were followed by the desorption step carried out in the headspace vials placed in the autosampler of the IMS device. Then, 200 μL of headspace was injected into the GC column coupled to the IMS for its analysis. The method was fully validated in terms of sensitivity, precision, and recovery. The LODs and LOQs obtained, when analytes were dissolved in saliva samples to consider the matrix effect, were within the range of 0.38–0.49 and 1.26–1.66 μg mL −1 , respectively. The relative standard deviations were 〈3.5 % for retention time and drift time values, which indicate that the method proposed can be applied to determine toxic compounds in saliva samples. Graphical abstract Summary of steps followed in the experimental set up of this work

Description: Accurate and precise glucose measurements are requisite for ensuring appropriate diagnosis and management of diseases related to hyperglycemia or hypoglycemia. It is necessary to have a higher order method to provide an accuracy base to which routine methods can be compared. We developed and evaluated a highly reliable measurement procedure based on isotope dilution liquid chromatography–tandem mass spectrometry (ID LC-MS/MS) with a simple one-step derivatization. An appropriate amount of serum was accurately weighed and spiked with an isotope-labeled internal standard. After protein precipitation, the supernatant was reacted with 1-phenyl-3-methyl-5-pyrazolone for chemical structural transformation. The glucose derivatives were analyzed with LC-MS/MS in positive electrospray ionization mode. The within-run and total CVs ranged from 0.28 to 0.42 % and from 0.42 to 0.76 %, respectively, for a concentration range of 1.691 to 15.676 mmol/L. A regression comparison of the presented method to an existing RMP based on ID GC-MS showed agreement with no statistical difference ( Y  = 0.9985X-0.008; 95 % CI for the slope, 0.9966 to 1.001; 95 % CI for the intercept, −0.012 to 0.019). The structural analogs of glucose with a molecular mass of 180 were tested, and no significant interference effect was found. The limit of quantification was estimated to 0.8 ng glucose in absolute amount. This method is accurate, simple, and can serve as a candidate reference measurement procedure (RMP) in the establishment of a serum glucose reference system.

Description: A standardized workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI imaging MS) is a prerequisite for the routine use of this promising technology in clinical applications. We present an approach to develop standard operating procedures for MALDI imaging MS sample preparation of formalin-fixed and paraffin-embedded (FFPE) tissue sections based on a novel quantitative measure of dataset quality. To cover many parts of the complex workflow and simultaneously test several parameters, experiments were planned according to a fractional factorial design of experiments (DoE). The effect of ten different experiment parameters was investigated in two distinct DoE sets, each consisting of eight experiments. FFPE rat brain sections were used as standard material because of low biological variance. The mean peak intensity and a recently proposed spatial complexity measure were calculated for a list of 26 predefined peptides obtained by in silico digestion of five different proteins and served as quality criteria. A five-way analysis of variance (ANOVA) was applied on the final scores to retrieve a ranking of experiment parameters with increasing impact on data variance. Graphical abstract MALDI imaging experiments were planned according to fractional factorial design of experiments for the parameters under study. Selected peptide images were evaluated by the chosen quality metric (structure and intensity for a given peak list), and the calculated values were used as an input for the ANOVA. The parameters with the highest impact on the quality were deduced and SOPs recommended.

Description: Polymers for recovery/removal of the antimicrobial agent oxytetracycline (OTC) from aqueous media were developed with use of computational design and molecular imprinting. 2-Hydroxyethyl methacrylate, 2-acrylamide-2-methylpropane sulfonic acid (AMPS), and mixtures of the two were chosen according to their predicted affinity for OTC and evaluated as functional monomers in molecularly imprinted polymers and nonimprinted polymers. Two levels of AMPS were tested. After bulk polymerization, the polymers were crushed into particles (200–1000 μm). Pressurized liquid extraction was implemented for template removal with a low amount of methanol (less than 20 mL in each extraction) and a few extractions (12–18 for each polymer) in a short period (20 min per extraction). Particle size distribution, microporous structure, and capacity to rebind OTC from aqueous media were evaluated. Adsorption isotherms obtained from OTC solutions (30–110 mg L -1 ) revealed that the polymers prepared with AMPS had the highest affinity for OTC. The uptake capacity depended on the ionic strength as follows: purified water 〉 saline solution (0.9 % NaCl) 〉 seawater (3.5 % NaCl). Polymer particles containing AMPS as a functional monomer showed a remarkable ability to clean water contaminated with OTC. The usefulness of the stationary phase developed for molecularly imprinted solid-phase extraction was also demonstrated. Graphical Abstract Selection of functional monomers by molecular modeling renders polymer networks suitable for removal of pollutants from contaminated aqueous environments, under either dynamic or static conditions.

Description: s Although PPL-based solid-phase extraction (SPE) has been widely used before dissolved organic matter (DOM) analyses via advanced measurements such as ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), much is still unknown about the structural and compositional changes in DOM pool through SPE. In this study, selected DOM from various sources were tested to elucidate the differences between before and after the SPE utilizing multiple analytical tools including fluorescence spectroscopy, FT-ICR-MS, and size exclusion chromatography with organic carbon detector (SEC-OCD). The changes of specific UV absorbance indicated the decrease of aromaticity after the SPE, suggesting a preferential exclusion of aromatic DOM structures, which was also confirmed by the substantial reduction of fluorescent DOM (FDOM). Furthermore, SEC-OCD results exhibited very low recoveries (1–9 %) for the biopolymer fraction, implying that PPL needs to be used cautiously in SPE sorbent materials for treating high molecular weight compounds (i.e., polysaccharides, proteins, and amino sugars). A careful examination via FT-ICR-MS revealed that the formulas lost by the SPE might be all DOM source-dependent. Nevertheless, the dominant missing compound groups were identified to be the tannins group with high O/C ratios (〉0.7), lignins/carboxyl-rich alicyclic molecules (CRAM), aliphatics with high H/C 〉1.5, and heteroatomic formulas, all of which were prevailed by pseudo-analogous molecular formula families with different methylene (−CH 2 ) units. Our findings shed new light on potential changes in the compound composition and the molecular weight of DOM upon the SPE, implying precautions needed for data interpretation. Graphical Abstract Tracking the characteristics of DOM from various origins upon PPL-based SPE utilizing EEMPARAFAC, SEC-OCD, and FT-ICR-MS

Description: Touch spray-mass spectrometry (TS-MS) is an ambient ionization technique (ionization of unprocessed samples in the open air) that may find intraoperative applications in quickly identifying the disease state of cancerous tissues and in defining surgical margins. In this study, TS-MS was performed on fresh kidney tissue (∼1–5 cm 3 ), within 1 h of resection, from 21 human subjects afflicted by renal cell carcinoma (RCC). The preliminary diagnostic value of TS-MS data taken from freshly resected tissue was evaluated. Principal component analysis (PCA) of the negative ion mode ( m / z 700–1000) data provided the separation between RCC (16 samples) and healthy renal tissue (13 samples). Linear discriminant analysis (LDA) on the PCA-compressed data estimated sensitivity (true positive rate) and specificity (true negative rate) of 98 and 95 %, respectively, based on histopathological evaluation. The results indicate that TS-MS might provide rapid diagnostic information in spite of the complexity of unprocessed kidney tissue and the presence of interferences such as urine and blood. Desorption electrospray ionization-MS imaging (DESI-MSI) in the negative ionization mode was performed on the tissue specimens after TS-MS analysis as a reference method. The DESI imaging experiments provided phospholipid profiles ( m / z 700–1000) that also separated RCC and healthy tissue in the PCA space, with PCA-LDA sensitivity and specificity of 100 and 89 %, respectively. The TS and DESI loading plots indicated that different ions contributed most to the separation of RCC from healthy renal tissue ( m / z 794 [PC 34:1 + Cl] − and 844 [PC 38:4 + Cl] − for TS vs. m / z 788 [PS 36:1 − H] − and 810 [PS 38:4 − H] − for DESI), while m / z 885 ([PI 38:4 − H] − ) was important in both TS and DESI. The prospect, remaining hurdles, and future work required for translating TS-MS into a method of intraoperative tissue diagnosis are discussed. Graphical abstract Touch spray-mass spectrometry used for lipid profiling of fresh human renal cell carcinoma. Left) Photograph of the touch spray probe pointed at the MS inlet. Right) Average mass spectra of healthy renal tissue (blue) and RCC (red)

Description: An innovative and effective extraction procedure based on molecularly imprinted solid-phase extraction (MISPE) was developed for the isolation of gonyautoxins 2,3 (GTX2,3) from Alexandrium minutum sample. Molecularly imprinted polymer microspheres were prepared by suspension polymerization and and were employed as sorbents for the solid-phase extraction of GTX2,3. An off-line MISPE protocol was optimized. Subsequently, the extract samples from A. minutum were analyzed. The results showed that the interference matrices in the extract were obviously cleaned up by MISPE procedures. This outcome enabled the direct extraction of GTX2,3 in A. minutum samples with extraction efficiency as high as 83 %, rather significantly, without any need for a cleanup step prior to the extraction. Furthermore, computational approach also provided direct evidences of the high selective isolation of GTX2,3 from the microalgal extracts.

Description: Aflatoxin M 1 (AFM 1 ), one of the most toxic mycotoxins, imposes serious health hazards. AFM 1 had previously been classified as a group 2B carcinogen [ 1 ] and has been classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) [ 2 ]. Determination of AFM 1 thus plays an important role for quality control of food safety. In this work, a sensitive and reliable aptasensor was developed for the detection of AFM 1 . The immobilization of aptamer through a strong interaction with biotin–streptavidin was used as a molecular recognition element, and its complementary ssDNA was employed as the template for a real-time quantitative polymerase chain reaction (RT-qPCR) amplification. Under optimized assay conditions, a linear relationship (ranging from 1.0 × 10 −4 to 1.0 μg L −1 ) was achieved with a limit of detection (LOD) down to 0.03 ng L −1 . In addition, the aptasensor developed here exhibits high selectivity for AFM 1 over other mycotoxins and small effects from cross-reaction with structural analogs. The method proposed here has been successfully applied to quantitative determination of AFM 1 in infant rice cereal and infant milk powder samples. Results demonstrated that the current approach is potentially useful for food safety analysis, and it could be extended to a large number of targets.

Description: This study aimed to rapidly determine 13 representative sulfonamide (SA) residues in pork by using a surface-modified hydrophilic polystyrene sulfonic acid (PSSA) electrospun nanofibrous membrane as the solid-phase extraction (SPE) pretreatment sorbent, followed by ultra-performance liquid chromatography (UPLC) analysis. The highly hydrophilic nature of PSSA nanofibrous membrane created by vacuum plasma treatment was characterized using Fourier transform infrared spectroscopy (FT-IR), field emission scanning electron microscopy (FE-SEM), water contact angle, and X-ray photoelectron spectroscopy (XPS) measurements. In the pretreatment procedures, 13 SA standards, which were spiked in the fatty pork samples, were extracted, enriched, and purified by the SPE procedure based on the principle of ion exchange with the sulfonic groups on the PSSA chains. Under the optimized conditions, the calibration curves of 13 SA compounds showed good linearities with correlation coefficients ( r ) of more than 0.99 in the range of 50.0–200 μg kg −1 . The mean recoveries of 13 SAs at the spiked concentrations of 50, 100, and 200 μg kg −1 were in the range of 70.3–92.5 % with average RSDs ( n = 6) of less than 15 % (except for sulfacetamide, 56.9–61.6 %). Compared with other pretreatment methods reported previously, less organic solvent (especially without degreasing the extract with n -hexane) was used in this time-saving SPE procedure, which avoids the possibility of emulsification and therefore enhances the recoveries. The developed and validated analysis method was sensitive, accurate, rapid, convenient, environmentally friendly, and was successfully applied for the detection of 13 SA residues in commercially available pork samples.