Showing papers in "Analytical and Bioanalytical Chemistry in 2013"

Molecular characterization of dissolved organic matter (DOM): a critical review

Abstract: Advances in water chemistry in the last decade have improved our knowledge about the genesis, com- position, and structure of dissolved organic matter, and its effect on the environment. Improvements in analyti- cal technology, for example Fourier-transform ion cyclo- tron (FT-ICR) mass spectrometry (MS), homo and hetero-correlated multidimensional nuclear magnetic res- onance (NMR) spectroscopy, and excitation emission matrix fluorimetry (EEMF) with parallel factor (PARAFAC) analysis for UV-fluorescence spectroscopy have resulted in these advances. Improved purification methods, for example ultrafiltration and reverse osmosis, have enabled facile desalting and concentration of fresh- ly collected DOM samples, thereby complementing the analytical process. Although its molecular weight (MW) remains undefined, DOM is described as a complex mixture of low-MW substances and larger-MW biomo- lecules, for example proteins, polysaccharides, and exo- cellular macromolecules. There is a general consensus that marine DOM originates from terrestrial and marine sources. A combination of diagenetic and microbial processes contributes to its origin, resulting in refractory organic matter which acts as carbon sink in the ocean. Ocean DOM is derived partially from humified products of plants decay dissolved in fresh water and transported to the ocean, and partially from proteinaceous and poly- saccharide material from phytoplankton metabolism, which undergoes in-situ microbial processes, becoming refractory. Some of the DOM interacts with radiation and is, therefore, defined as chromophoric DOM (CDOM). CDOM is classified as terrestrial, marine, anthropogenic, or mixed, depending on its origin. Ter- restrial CDOM reaches the oceans via estuaries, whereas autochthonous CDOM is formed in sea water by micro- bial activity; anthropogenic CDOM is a result of human activity. CDOM also affects the quality of water, by shielding it from solar radiation, and constitutes a car- bon sink pool. Evidence in support of the hypothesis that part of marine DOM is of terrestrial origin, being the result of a long-term carbon sedimentation, has been obtained from several studies discussed herein.

Sensing approaches on paper-based devices: a review

Abstract: Paper has been present in the world of analytical chemistry for centuries, but it seems that just a few years back it was rediscovered as a valuable substrate for sensors. We can easily list some of the countless advantages of this simple cellulosic substrate, including mechanical properties, three-dimensional fibrous structure, biocompatibility and biodegradability, easiness of production and modification, reasonable price, and availability all over the world. Those characteristics make paper a first-choice substrate for disposable sensors and integrated sensing platforms. Nowadays, numerous examples of paper-based sensors are being presented in the literature. This review describes some of the most prominent examples classifying them by type of detection: optical (colorimetric, fluorescence, surface-enhanced Raman spectroscopy, and transmittance methods) and electrochemical (voltammetric, potentiometric, and conductivity-based methods). We take a closer look at recent advances in immunoassays fabricated on paper, excluding simple lateral flow tests assembled on nitrocellulose. This review also summarizes the main advantages and disadvantages of the use of paper as a substrate for sensors, as well as its impact on their performance and application, presents a short history of paper in analytical chemistry, and discusses fabrication methods and available sources of paper.

New trends in the electrochemical sensing of dopamine.

Abstract: Since the early 70s electrochemistry has been used as a powerful analytical technique for monitoring electroactive species in living organisms. In particular, after extremely rapid evolution of new micro and nanotechnology it has been established as an invaluable technique ranging from experiments in vivo to measurement of exocytosis during communication between cells under in vitro conditions. This review highlights recent advances in the development of electrochemical sensors for selective sensing of one of the most important neurotransmitters—dopamine. Dopamine is an electroactive catecholamine neurotransmitter, abundant in the mammalian central nervous system, affecting both cognitive and behavioral functions of living organisms. We have not attempted to cover a large time-span nor to be comprehensive in presenting the vast literature devoted to electrochemical dopamine sensing. Instead, we have focused on the last five years, describing recent progress as well as showing some problems and directions for future development.

Recent advances in the development of graphene-based surface plasmon resonance (SPR) interfaces

Abstract: Surface plasmon resonance (SPR) is a powerful technique for measurement of biomolecular interactions in real-time in a label-free environment. One of the most common techniques for plasmon excitation is the Kretschmann configuration, and numerous studies of ligand–analyte interactions have been performed on surfaces functionalized with a variety of biomolecules, for example DNA, RNA, glycans, proteins, and peptides. A significant limitation of SPR is that the substrate must be a thin metal film. Post-coating of the metal thin film with a thin dielectric top layer has been reported to enhance the performance of the SPR sensor, but is highly dependent on the thickness of the upper layer and its dielectric constant. Graphene is a single-atom thin planar sheet of sp2 carbon atoms perfectly arranged in a honeycomb lattice. Graphene and graphene oxide are good supports for biomolecules because of their large surface area and rich π conjugation structure, making them suitable dielectric top layers for SPR sensing. In this paper, we review some of the key issues in the development of graphene-based SPR chips. The actual challenges of using these interfaces for studying biomolecular interactions will be discussed and the first examples of the use of graphene-on-metal SPR interfaces for biological sensing will be presented.

Application of quality by design to the development of analytical separation methods

Abstract: Recent pharmaceutical regulatory documents have stressed the critical importance of applying quality by design (QbD) principles for in-depth process understanding to ensure that product quality is built in by design. This article outlines the application of QbD concepts to the development of analytical separation methods, for example chromatography and capillary electrophoresis. QbD tools, for example risk assessment and design of experiments, enable enhanced quality to be integrated into the analytical method, enabling earlier understanding and identification of variables affecting method performance. A QbD guide is described, from identification of quality target product profile to definition of control strategy, emphasizing the main differences from the traditional quality by testing (QbT) approach. The different ways several authors have treated single QbD steps of method development are reviewed and compared. In a final section on outlook, attention is focused on general issues which have arisen from the surveyed literature, and on the need to change the researcher’s mindset from the QbT to QbD approach as an important analytical trend for the near future.

Detection and characterization of silver nanoparticles in chicken meat by asymmetric flow field flow fractionation with detection by conventional or single particle ICP-MS

Abstract: A method of analysis of silver nanoparticles (AgNPs) in chicken meat was developed. The homogenized chicken meat sample, which was spiked with AgNPs, was subjected to enzymolysis by Proteinase K for 40 min at 37 °C. Transmission electron microscopy and inductively coupled plasma mass spectrometry (ICP-MS) in single particle mode were used to characterize the number-based size distribution of AgNPs in the meat digestate. Because similar size distributions were found in the meat digestate and in the aqueous suspension of AgNPs used for spiking the meat, it was shown that no detectable dissolution of the AgNPs took place during the sample preparation stage. The digestate was injected into the asymmetric flow field flow fractionation (AF4) -ICP-MS system, which enabled fractionation of nanoparticles from the remaining meat matrix, and resulted in one large peak in the fractograms as well as two smaller peaks eluting close to the void volume. The recovery of silver contained in the large AgNP peak was around 80 %. Size determination of AgNPs in the meat matrix, based on external size calibration of the AF4 channel, was hampered by non-ideal (early elution) behavior of the AgNPs. Single particle ICP-MS was applied for determination of the number-based particle size distribution of AgNPs in collected fractions. The presented work describes for the first time the coupling of AF4 and ICP-MS for AgNP separation in a food matrix.

Lateral-flow immunoassays for mycotoxins and phycotoxins: a review

Abstract: Natural toxin (for example mycotoxin and phycotoxin) contamination of food is of safety and economic concern, so much effort is devoted to the development of screening methods which enable the toxins to be continuously and widely monitored in food and feed. More generally speaking, rapid and non-instrumental assays for detection of a variety of food contaminants are generating ever-increasing scientific and technological interest because they enable high-throughput, economical, on-site monitoring of such contaminants. Among rapid methods for first-level screening of food contaminants, lateral-flow immunoassay (LFIA), also named immunochromatographic assay or immune-gold colloid immunoassay, has recently attracted scientific and industrial interest because of its attractive property of enabling very rapid, one-step, in-situ analysis. This review focuses on new aspects of the development and optimization of lateral-flow devices for mycotoxin and phycotoxin detection, including strategies for management of matrix interference and, particularly, for investigation of the improvements achieved by signal-enhancing strategies or by application of non-gold nanoparticle signal reporters.

Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk.

Abstract: A novel rapid (20 min) fluorescent lateral flow test for chloramphenicol (CAP) detection in milk was developed. The chosen format is a binding-inhibition assay. Water-soluble quantum dots with an emission peak at 625 nm were applied as a label. Milk samples were diluted by 20 % with phosphate buffer to eliminate the matrix effect. The result of the assay could be seen by eye under UV light excitation or registered by a portable power-dependent photometer. The limit of CAP detection by the second approach is 0.2 ng/mL, and the limit of quantitation is 0.3 ng/mL.

Normalization procedures and reference material selection in stable HCNOS isotope analyses: an overview

Abstract: The uncertainties of stable isotope results depend not only on the technical aspects of measurements, but also on how raw data are normalized to one of the international isotope scales. The inconsistency in the normalization methods used and in the selection of standards may lead to substantial differences in the results obtained. Therefore, unification of the data processing protocols employed is highly desirable. The best performing methods are two-point or multipoint normalization methods based on linear regression. Linear regression is most robust when based on standards that cover the entire range of δ values typically observed in nature, regardless of the δ values of the samples analysed. The uncertainty can be reduced by 50 % if measurements of two different standards are performed four times, or measurements of four standards are performed twice, with each batch of samples. Chemical matrix matching between standards and samples seems to be critical for δ 18O of nitrate or δ 2H of hair samples (thermal conversion/elemental analyser), for example; however, it is not necessarily always critical for all types of samples and techniques (e.g. not for most δ 15N and δ 13C elemental analyser analyses). To ensure that all published data can be recalculated, if δ values of standards or the isotope scales are to be updated, the details of the normalization technique and the δ values of the standards used should always be clearly reported.

Inkjet printed (bio)chemical sensing devices

Abstract: Inkjet printing has evolved from an office printing application to become an important tool in industrial mass fabrication. In parallel, this technology is increasingly used in research laboratories around the world for the fabrication of entire (bio)chemical sensing devices or single functional elements of such devices. Regularly stated characteristics of inkjet printing making it attractive to replace an alternative material deposition method are low cost, simplicity, high resolution, speed, reproducibility, flexibility, non-contact, and low amount of waste generated. With this review, we give an overview over areas of (bio)chemical sensing device development profiting from inkjet printing applications. A variety of printable functional sensor elements are introduced by examples, and the advantages and challenges of the inkjet method are pointed out. It is demonstrated that inkjet printing is already a routine tool for the fabrication of some (bio)chemical sensing devices, but also that novel applications are being continuously developed. Finally, some inherent limitations of the method and challenges for the further exploitation of this technology are pointed out.

Development and validation of a (semi-)quantitative UHPLC-MS/ MS method for the determination of 191 mycotoxins and other fungal metabolites in almonds, hazelnuts, peanuts and pistachios

Abstract: A multi-target method for the determination of 191 fungal metabolites in almonds, hazelnuts, peanuts and pistachios was developed. The method includes all mycotoxins regulated in the European Union and mycotoxins regularly found in food. After extraction with an acidified acetonitrile water mixture, the raw extract was diluted and injected directly into the UHPLC-MS/MS system. In two chromatographic runs, analysis was performed in positive and in negative ionisation mode. The method was in-house validated for the most important 65 analytes in these four commodities. Apparent recoveries between 80 and 120 % were obtained for about half of the analyte–matrix combinations. Good repeatabilities (standard deviations < 10 %) were achieved for the vast majority (83 %) of all cases. Only in 6 % of all combinations did the standard deviations exceed 15 %. Matrix effects, arising during electrospray ionisation, significantly influenced the determination. For instance, signal suppression was observed for several early-eluting analytes and also signal enhancement up to 295 % for physcion in peanuts was determined. Concerning extraction recovery, 94 % of the analyte–matrix combinations showed values higher than 50 %. Lower limits of quantification ranged between 0.04 μg kg−1 for enniatin B3 in peanuts and 500 μg kg−1 for HC toxin in hazelnuts. Additionally, the applicability of the developed method was demonstrated through the analysis of 53 naturally contaminated nut samples from Austria and Turkey. Overall, 40 toxins were quantified; the most frequently found mycotoxins were beauvericin (79 %), enniatin B (62 %) and macrosporin (57 %). In the most contaminated hazelnut sample, 26 different fungal metabolites were detected.

Analytical methods for the determination of halogens in bioanalytical sciences: a review

Abstract: Fluorine, chlorine, bromine, and iodine have been studied in biological samples and other related matrices owing to the need to understand the biochemical effects in living organisms. In this review, the works published in last 20 years are covered, and the main topics related to sample preparation methods and analytical techniques commonly used for fluorine, chlorine, bromine, and iodine determination in biological samples, food, drugs, and plants used as food or with medical applications are discussed. The commonest sample preparation methods, as extraction and decomposition using combustion and pyrohydrolysis, are reviewed, as well as spectrometric and electroanalytical techniques, spectrophotometry, total reflection X-ray fluorescence, neutron activation analysis, and separation systems using chromatography and electrophoresis. On this aspect, the main analytical challenges and drawbacks are highlighted. A discussion related to the availability of certified reference materials for evaluation of accuracy is also included, as well as a discussion of the official methods used as references for the determination of halogens in the samples covered in this review.

Affinity monolith chromatography: a review of principles and recent analytical applications

Abstract: Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis, or study of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments and applications of this method, with particular emphasis being given to work that has appeared in the last 5 years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths, and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns, and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal ions, and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized-metal-ion affinity chromatography, dye–ligand affinity chromatography, chiral separations, and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing, and biotechnology. Current trends and possible directions in AMC are also discussed.

Characterising and correcting batch variation in an automated direct infusion mass spectrometry (DIMS) metabolomics workflow

Abstract: Direct infusion mass spectrometry (DIMS)-based untargeted metabolomics measures many hundreds of metabolites in a single experiment. While every effort is made to reduce within-experiment analytical variation in untargeted metabolomics, unavoidable sources of measurement error are introduced. This is particularly true for large-scale multi-batch experiments, necessitating the development of robust workflows that minimise batch-to-batch variation. Here, we conducted a purpose-designed, eight-batch DIMS metabolomics study using nanoelectrospray (nESI) Fourier transform ion cyclotron resonance mass spectrometric analyses of mammalian heart extracts. First, we characterised the intrinsic analytical variation of this approach to determine whether our existing workflows are fit for purpose when applied to a multi-batch investigation. Batch-to-batch variation was readily observed across the 7-day experiment, both in terms of its absolute measurement using quality control (QC) and biological replicate samples, as well as its adverse impact on our ability to discover significant metabolic information within the data. Subsequently, we developed and implemented a computational workflow that includes total-ion-current filtering, QC-robust spline batch correction and spectral cleaning, and provide conclusive evidence that this workflow reduces analytical variation and increases the proportion of significant peaks. We report an overall analytical precision of 15.9 %, measured as the median relative standard deviation (RSD) for the technical replicates of the biological samples, across eight batches and 7 days of measurements. When compared against the FDA guidelines for biomarker studies, which specify an RSD of <20 % as an acceptable level of precision, we conclude that our new workflows are fit for purpose for large-scale, high-throughput nESI DIMS metabolomics studies.

A high-sensitivity ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method for screening synthetic cannabinoids and other drugs of abuse in urine

Abstract: The continuing emergence of designer drugs imposes high demands on the scope and sensitivity of toxicological drug screening procedures. An ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method was developed for screening and simultaneous confirmation of both designer drugs and other drugs of abuse in urine samples in a single run. The method covered selected synthetic cannabinoids and cathinones, amphetamines, natural cannabinoids, opioids, cocaine and other important drugs of abuse, together with their main urinary metabolites. The database consisted of 277 compounds with molecular formula and exact monoisotopic mass; retention time was included for 192 compounds, and primary and secondary qualifier ion exact mass for 191 and 95 compounds, respectively. Following a solid-phase extraction, separation was performed by UHPLC and mass analysis by HR-TOFMS. MS, and broad-band collision-induced dissociation data were acquired at m/z range 50-700. Compound identification was based on a reverse database search with acceptance criteria for retention time, precursor ion mass accuracy, isotopic pattern and abundance of qualifier ions. Mass resolving power in spiked urine samples was on average FWHM 23,500 and mass accuracy 0.3 mDa. The mean and median cut-off concentrations determined for 75 compounds were 4.2 and 1 ng/mL, respectively. The range of cut-off concentrations for synthetic cannabinoids was 0.2-60 ng/mL and for cathinones 0.7-15 ng/mL. The method proved to combine high sensitivity and a wide scope in a manner not previously reported in drugs of abuse screening. The method's feasibility was demonstrated with 50 authentic urine samples.

High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-carbon metabolism using HPLC-MS/MS

Abstract: Risk of chronic diseases, like cardiovascular disease and cancer, has been associated with biomarkers related to one-carbon metabolism, which comprises a metabolic network of cross-talking pathways. To address this complexity in epidemiological studies, we have established an isotope dilution HPLC-MS/MS method for quantification of 12 biomarkers and metabolites. All sample handling is performed by a robotic workstation. The assay uses 45 μL of plasma, and sample treatment consists of protein precipitation by trichloroacetic acid. The analytes were separated on a Fortis Phenyl column using an isocratic mobile phase that contained water, methanol and acetic acid. Methionine, methionine sulfoxide, choline, betaine, dimethylglycine, arginine, asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine, creatinine, cystathionine and trimethyllysine all showed limits of detection well below the 5th percentile of plasma distributions in healthy humans, coefficients of variation were in the range 2.2-12.3 %, and recoveries were 80-131 %. Simple sample processing, low-volume consumption, multiplexing and high capacity/short run time of this method make it suitable for large-scale metabolic profiling of precious biobank samples.

Development of a UPLC-MS/MS method for the determination of ten anticancer drugs in hospital and urban wastewaters, and its application for the screening of human metabolites assisted by information-dependent acquisition tool (IDA) in sewage samples

Abstract: In the present work, the development, optimization, and validation (including a whole stability study) of a fast, reliable, and comprehensive method for the analysis of ten anticancer drugs in hospital and urban wastewater is described. Extraction of these pharmaceutical compounds was performed using automated off-line solid-phase extraction followed by their determination by ultra-performance liquid chromatography coupled to a triple quadrupole–linear ion trap mass spectrometer. Target compounds include nine cytotoxic agents: cyclophosphamide, ifosfamide, docetaxel, paclitaxel, etoposide, vincristine, tamoxifen, methotrexate, and azathioprine; and the cytotoxic quinolone, ciprofloxacin. Method detection limits (MDL) ranged from 0.8 to 24 ng/L. Levels found of cytostatic agents in the hospital and wastewater influents did not differ significantly, and therefore, hospitals cannot be considered as the primary source of this type of contaminants. All the target compounds were detected in at least one of the influent samples analyzed: Ciprofloxacin, cyclophosphamide, tamoxifen, and azathioprine were found in most of them and achieving maximum levels of 14.725, 0.201, 0.133, and 0.188 μg/L, respectively. The rest of target cancer drugs were less frequently detected and at values ranging between MDL and 0.406 μg/L. Furthermore, a feasible, useful, and advantageous approach based on information acquisition tool (information-dependent acquisition) was used for the screening of human metabolites in hospital effluents, where the hydroxy tamoxifen, endoxifen, and carboxyphosphamide were detected.

Sweeteners from plants—with emphasis on Stevia rebaudiana (Bertoni) and Siraitia grosvenorii (Swingle)

Abstract: In addition to their widely recognized use as dietary supplement ingredients, plant-derived compounds are increasingly used as natural sweeteners. The search for nonnutritive sweeteners has been stimulated over the last 20-30 years by concern over demonstrated or suspected relationships between consumption of sucrose and high-fructose corn syrups and a variety of health-related conditions. In the USA, there is increased use of plant extracts known to contain highly sweet terpenoids. Purified extracts of Stevia rebaudiana (Bertoni) containing the diterpene glycosides stevioside and rebaudioside A are popular as sweeteners and are also used as dietary supplements, and soft drinks and nutritional and energy shakes incorporating extracts of Siraitia grosvenorii (Swingle) fruits containing sweet triterpene glycosides such as mogroside V are also on the market. Here, we review recent studies on these two important sources of noncaloric natural sweeteners, including analytical methods used to identify and quantify specific constituents and structural features relating to their sweetness. We also review the generally recognized as safe status of specific components and their status with respect to review by the Joint FAO/WHO Expert Committee on Food Additives.

Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay.

Abstract: MicroRNAs (miRNAs, miRs) are naturally occurring small RNAs (approximately 22 nucleotides in length) that have critical functions in a variety of biological processes, including tumorigenesis. They are an important target for detection technology for future medical diagnostics. In this paper we report an electrochemical method for miRNA detection based on paramagnetic beads and enzyme amplification. In particular, miR 222 was chosen as model sequence, because of its involvement in brain, lung, and liver cancers. The proposed bioassay is based on biotinylated DNA capture probes immobilized on streptavidin-coated paramagnetic beads. Total RNA was extracted from the cell sample, enriched for small RNA, biotinylated, and then hybridized with the capture probe on the beads. The beads were then incubated with streptavidin-alkaline phosphatase and exposed to the appropriate enzymatic substrate. The product of the enzymatic reaction was electrochemically monitored. The assay was finally tested with a compact microfluidic device which enables multiplexed analysis of eight different samples with a detection limit of 7 pmol L(-1) and RSD = 15 %. RNA samples from non-small-cell lung cancer and glioblastoma cell lines were also analyzed.

Simultaneous quantification of 28 synthetic cathinones and metabolites in urine by liquid chromatography-high resolution mass spectrometry

Abstract: Synthetic cathinones are novel stimulants derived from cathinone, with amphetamines or cocaine-like effects, often labeled “not for human consumption” and considered “legal highs”. Emergence of these new designer drugs complicate interpretation of forensic and clinical cases, with introduction of many new analogs designed to circumvent legislation and vary effects and potencies. We developed a method for the simultaneous quantification of 28 synthetic cathinones, including four metabolites, in urine by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). These cathinones include cathinone, methcathinone, and synthetic cathinones position-3’-substituted, N-alkyl-substituted, ring-substituted, methylenedioxy-substituted, and pyrrolidinyl-substituted. One mL phosphate buffer pH 6 and 25 μL IStd solution were combined with 0.25 mL urine, and subjected to solid phase cation exchange extraction (SOLA SCX). The chromatographic reverse-phase separation was achieved with a gradient mobile phase of 0.1 % formic acid in water and in acetonitrile in 20 min. We employed a Q Exactive high resolution mass spectrometer, with compounds identified and quantified by target-MSMS experiments. The assay was linear from 0.5–1 to 100 μg/L, with limits of detection of 0.25–1 μg/L. Imprecision (n = 20) was <15.9 % and accuracy (n = 20) 85.2–118.1 %. Extraction efficiency was 78.9–116.7 % (CV 1.4–16.7 %, n = 5), process efficiency 57.7–104.9 %, and matrix effects from −29.5 % to 1.5 % (CV 1.9–13.1 %, n = 10). Most synthetic cathinones were stable at 4 °C for 72 h (n = 27) and after 3 freeze-thaw cycles (n = 26), but many (n = 19) were not stable at room temperature for 24 h (losses up to −67.6 %). The method was applied to authentic urine specimens from synthetic cathinone users. This method provides a comprehensive confirmation method for 28 synthetic cathinones in urine, with good selectivity and specificity.

Cellobiose dehydrogenase modified electrodes: advances by materials science and biochemical engineering

Abstract: The flavocytochrome cellobiose dehydrogenase (CDH) is a versatile biorecognition element capable of detecting carbohydrates as well as quinones and catecholamines. In addition, it can be used as an anode biocatalyst for enzymatic biofuel cells to power miniaturised sensor–transmitter systems. Various electrode materials and designs have been tested in the past decade to utilize and enhance the direct electron transfer (DET) from the enzyme to the electrode. Additionally, mediated electron transfer (MET) approaches via soluble redox mediators and redox polymers have been pursued. Biosensors for cellobiose, lactose and glucose determination are based on CDH from different fungal producers, which show differences with respect to substrate specificity, pH optima, DET efficiency and surface binding affinity. Biosensors for the detection of quinones and catecholamines can use carbohydrates for analyte regeneration and signal amplification. This review discusses different approaches to enhance the sensitivity and selectivity of CDH-based biosensors, which focus on (1) more efficient DET on chemically modified or nanostructured electrodes, (2) the synthesis of custom-made redox polymers for higher MET currents and (3) the engineering of enzymes and reaction pathways. Combination of these strategies will enable the design of sensitive and selective CDH-based biosensors with reduced electrode size for the detection of analytes in continuous on-site and point-of-care applications.

Optimization of harvesting, extraction, and analytical protocols for UPLC-ESI-MS-based metabolomic analysis of adherent mammalian cancer cells.

Abstract: In this study, a liquid chromatography mass spectrometry (LC/MS)-based metabolomics protocol was optimized for quenching, harvesting, and extraction of metabolites from the human pancreatic cancer cell line Panc-1. Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in water were compared for sample harvesting. Four different extraction methods were compared to investigate the efficiency of intracellular metabolite extraction, including pure acetonitrile, methanol, methanol/chloroform/H2O, and methanol/chloroform/acetonitrile. The separation efficiencies of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) with UPLC-QTOF-MS were also evaluated. Global metabolomics profiles were compared; the number of total detected features and the recovery and relative extraction efficiencies of target metabolites were assessed. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Direct scraping after flash quenching with liquid nitrogen was chosen to harvest Panc-1 cells which allowed for samples to be stored before extraction. Methanol/chloroform/H2O was chosen as the optimal extraction solvent to recover the highest number of intracellular features with the best reproducibility. HILIC had better resolution for intracellular metabolites of Panc-1 cells. This optimized method therefore provides high sensitivity and reproducibility for a variety of cellular metabolites and can be applicable to further LC/MS-based global metabolomics study on Panc-1 cell lines and possibly other cancer cell lines with similar chemical and physical properties.

GC-MS-based urine metabolic profiling of autism spectrum disorders

Abstract: Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders resulting from multiple factors. Diagnosis is based on behavioural and developmental signs detected before 3 years of age, and there is no reliable biological marker. The purpose of this study was to evaluate the value of gas chromatography combined with mass spectroscopy (GC-MS) associated with multivariate statistical modeling to capture the global biochemical signature of autistic individuals. GC-MS urinary metabolic profiles of 26 autistic and 24 healthy children were obtained by liq/liq extraction, and were or were not subjected to an oximation step, and then were subjected to a persilylation step. These metabolic profiles were then processed by multivariate analysis, in particular orthogonal partial least-squares discriminant analysis (OPLS-DA, R(2)Y(cum) = 0.97, Q(2)(cum) = 0.88). Discriminating metabolites were identified. The relative concentrations of the succinate and glycolate were higher for autistic than healthy children, whereas those of hippurate, 3-hydroxyphenylacetate, vanillylhydracrylate, 3-hydroxyhippurate, 4-hydroxyphenyl-2-hydroxyacetate, 1H-indole-3-acetate, phosphate, palmitate, stearate, and 3-methyladipate were lower. Eight other metabolites, which were not identified but characterized by a retention time plus a quantifier and its qualifier ion masses, were found to differ between the two groups. Comparison of statistical models leads to the conclusion that the combination of data obtained from both derivatization techniques leads to the model best discriminating between autistic and healthy groups of children.

Increased sensitivity of lateral flow immunoassay for ochratoxin a through silver enhancement

Abstract: Silver nucleation on gold has been exploited for signal amplification and has found application in several qualitative and quantitative bio-sensing techniques, thanks to the simplicity of the method and the high sensitivity achieved. Very recently, this technique has been tentatively applied to improve the performance of gold-based immunoassays. In this work, the exploitation of the signal amplification due to silver deposition on gold nanoparticles has been first applied to a competitive lateral flow immunoassay (LFIA). The signal enhancement due to silver allowed us to strongly reduce the amount of the competitor and of specific antibodies employed to build an LF device for measuring ochratoxin A (OTA), thus permitting the attainment of a highly sensitive assessment of OTA contamination, with a sensitivity gain of more than 10-fold compared to the gold-based LFIA that used the same immunoreagents and to all previously reported LFIA for measuring OTA. In addition, a less sensitive "quantitative" LFIA could be established, by suitably tuning competitor and antibody amounts, which was characterized by reproducible and accurate OTA determinations (RSD% 6-12%, recovery% 82-117%). The quantitative system allowed a reliable OTA quantification in wines and grape musts at the microgram per liter level requested by the European legislation, as demonstrated by a highly results obtained through the quantitative silver-enhanced LFIA and a reference HPLC-FLD on 30 samples.

A microfluidic electrochemiluminescent device for detecting cancer biomarker proteins

Abstract: We describe an electrochemiluminescence (ECL) immunoarray incorporated into a prototype microfluidic device for highly sensitive protein detection and apply this system to accurate, sensitive measurements of prostate-specific antigen (PSA) and interleukin-6 (IL-6) in serum. The microfluidic system employed three molded polydimethylsiloxane (PDMS) channels on a conductive pyrolytic graphite chip (2.5 × 2.5 cm) inserted into a machined chamber and interfaced with a pump, switching valve, and sample injector. Each of the three PDMS channels encompasses three 3 μL analytical wells. Capture-antibody-decorated single-wall carbon nanotube forests are fabricated in the bottom of the wells. The antigen is captured by these antibodies on the well bottoms. Then, a RuBPY-silica-secondary antibody (Ab2) label is injected to bind to antigen on the array, followed by injection of sacrificial reductant tripropylamine (TPrA) to produce ECL. For detection, the chip is placed into an open-top ECL measuring cell, and the channels are in contact with electrolyte in the chamber. Potential applied at 0.95 V versus Ag/AgCl oxidizes TPrA to produce ECL by redox cycling the RuBPY species in the particles, and ECL light is measured by a charge-coupled device camera. This approach achieved ultralow detection limits of 100 fg mL(-1) for PSA (9 zeptomole) and 10 fg mL(-1) (1 zeptomole) for IL-6 in calf serum, a 10-25-fold improvement of a similar non-microfluidic array. PSA and IL-6 in synthetic cancer patient serum samples were detected in 1.1 h and results correlated well with single-protein enzyme-linked immunosorbent assays.

Determination of sulfite with emphasis on biosensing methods: a review

Abstract: Sulfite is used as a preservative in a variety of food and pharmaceutical industries to inhibit enzymatic and nonenzymatic browning and in brewing industries as an antibacterial and antioxidizing agent. Convenient and reproducible analytical methods employing sulfite oxidase are an attractive alternative to conventional detection methods. Sulfite biosensors are based on measurement of either O2 or electrons generated from splitting of H2O2 or heat released during oxidation of sulfite by immobilized sulfite oxidase. Sulfite biosensors can be grouped into 12 classes. They work optimally within 2 to 900 s, between pH 6.5 and 9.0, 25 and 40 °C, and in the range from 0 to 50,000 μM, with detection limit between 0.2 and 200 μM. Sulfite biosensors measure sulfite in food, beverages, and water and can be reused 100–300 times over a period of 1–240 days. The review presents the principles, merits, and demerits of various analytical methods for determination of sulfite, with special emphasis on sulfite biosensors.

Advances in pesticide biosensors: current status, challenges, and future perspectives.

Abstract: Public concern over pesticide residues has been increasing dramatically owing to the high toxicity and bioaccumulation effects of pesticides and the serious risks that they pose to the environment and human health. It is therefore crucial to monitor pesticide residues by using various analytical methods and techniques, especially highly sensitive, highly selective, simple, rapid, cost-effective, and portable ones. Biosensor strategies have become research hotspots and ideal candidates for pesticide detection, having such features as high sensitivity, fast response, robustness, low cost and miniaturization, as well as in situ and real-time monitoring. This review covers advances in the design and fabrication of biosensors for pesticide detection since 2005. Special emphasis is placed on the state-of-art selection of receptors, the use of different transduction techniques and fast screening strategies, and the application of various biosensors developed in food and environmental safety. Both advantages and drawbacks of these techniques are then summarized. Finally, challenges, strategies, and perspectives in further developing pesticide biosensors are also discussed.

Selection and identification of ssDNA aptamers recognizing zearalenone

Abstract: Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z31, with a dissociation constant (K d) of 41 ± 5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14 × 10−9 to 3.14 × 10−5 M for ZEN, and the detection limit was 7.85 × 10−10 M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products.

Stable isotopic labelling-assisted untargeted metabolic profiling reveals novel conjugates of the mycotoxin deoxynivalenol in wheat.

Abstract: An untargeted screening strategy for the detection of biotransformation products of xenobiotics using stable isotopic labelling (SIL) and liquid chromatography–high resolution mass spectrometry (LC-HRMS) is reported. The organism of interest is treated with a mixture of labelled and non-labelled precursor and samples are analysed by LC-HRMS. Raw data are processed with the recently developed MetExtract software for the automated extraction of corresponding peak pairs. The SIL-assisted approach is exemplified by the metabolisation of the Fusarium mycotoxin deoxynivalenol (DON) in planta. Flowering ears were inoculated with 100 μg of a 1 + 1 (v/v) mixture of non-labelled and fully labelled DON. Subsequent sample preparation, LC-HRMS measurements and data processing revealed a total of 57 corresponding peak pairs, which originated from ten metabolites. Besides the known DON and DON-3-glucoside, which were confirmed by measurement of authentic standards, eight further DON-biotransformation products were found by the untargeted screening approach. Based on a mass deviation of less than ±5 ppm and MS/MS measurements, one of these products was annotated as DON-glutathione (GSH) conjugate, which is described here for the first time for wheat. Our data further suggest that two DON-GSH-related metabolites, the processing products DON-S-cysteine and DON-S-cysteinyl-glycine and five unknown DON conjugates were formed in planta. Future MS/MS measurements shall reveal the molecular structures of the detected conjugates in more detail.

Microfluidic channel with embedded SERS 2D platform for the aptamer detection of ochratoxin A.

Abstract: A selective aptameric sequence is adsorbed on a two-dimensional nanostructured metallic platform optimized for surface-enhanced Raman spectroscopy (SERS) measurements. Using nanofabrication methods, a metallic nanostructure was prepared by electron-beam lithography onto a glass coverslip surface and embedded within a microfluidic channel made of polydimethylsiloxane, allowing one to monitor in situ SERS fingerprint spectra from the adsorbed molecules on the metallic nanostructures. The gold structure was designed so that its localized surface plasmon resonance matches the excitation wavelength used for the Raman measurement. This optofluidic device is then used to detect the presence of a toxin, namely ochratoxin-A (OTA), in a confined environment, using very small amounts of chemicals, and short data acquisition times, by taking advantage of the optical properties of a SERS platform to magnify the Raman signals of the aptameric monolayer system and avoiding chemical labeling of the aptamer or the OTA target.