Analytical and Bioanalytical Chemistry (v.408, #17)

Hollandaise challenge by Hervé This (4467-4468).

Solution to Mohr’s method challenge by Juris Meija; Anna Maria Michałowska-Kaczmarczyk; Tadeusz Michałowski (4469-4471).

The exceptional photophysical properties and the nanometric dimensions of colloidal semiconductor quantum dots (QD) have strongly attracted the bioanalytical community over the last approximately 20 y. In particular, the integration of QDs in the analysis of biological components and interactions, and the related diagnostics using Förster resonance energy transfer (FRET), have allowed researchers to significantly improve and diversify fluorescence-based biosensing. In this TRENDS article, we review some recent developments in QD-FRET biosensing that have implemented this technology in electronic consumer products, multiplexed analysis, and detection without light excitation for diagnostic applications. In selected examples of smartphone-based imaging, single- and multistep FRET, steady-state and time-resolved spectroscopy, and bio/chemiluminescence detection of QDs used as both FRET donors and acceptors, we highlight the advantages of QD-based FRET biosensing for multiplexed and sensitive diagnostics. Graphical Abstract Quantum dots (QDs) can be applied as donors and/or acceptors for Förster resonance energy transfer- (FRET-) based biosensing for multiplexed and sensitive diagnostics in various assay formats
Keywords: Fluorescence; Spectroscopy; Imaging; Multiplexing; Nanoparticle; Immunoassay; DNA; RNA; Smartphone; BRET; CRET

Gold nanoparticles (AuNPs) assisted laser desorption/ionization mass spectrometry (GALDI-MS) provided new horizons and offered many functions for various applications. This review summarized AuNPs applications for analytical, biotechnology and proteomics. AuNPs efficiently absorbed the laser radiation and transferred the energy to the analyte for the desorption/ionization process. The unique features of AuNPs such as large surface area and high absorption coefficient lead not only to high resolution, low interference and low limit of detection, but also offered selective detection for certain species. AuNPs provided an excellent surface for the analysis of several species such as small molecules, biomarkers, proteins and cells (pathogenic bacteria or cancer cells). AuNPs played many roles such as surface for LDI-MS, probe and stationary phase for separation or preconcentration. AuNPs modified various surface chemistry was applied for a wide range of different wavelength. AuNPs severed as a source of Au+ ions that were suitable for analyte cationisation. Characterization of Au nanoclusters (AuNCs) by mass spectrometry, pros and cons were also highlighted. Graphical Abstract Schematic representation of the analysis by Gold Nanoparticles Assisted Laser Desorption/Ionization Mass Spectrometry (GALDI-MS)
Keywords: Gold nanoparticles; Biosening; Proteomics; Biotechnology; Cancer cells; Mass spectrometry

Microfabricated, amperometric, enzyme-based biosensors for in vivo applications by Andreas Weltin; Jochen Kieninger; Gerald A. Urban (4503-4521).
Miniaturized electrochemical in vivo biosensors allow the measurement of fast extracellular dynamics of neurotransmitter and energy metabolism directly in the tissue. Enzyme-based amperometric biosensing is characterized by high specificity and precision as well as high spatial and temporal resolution. Aside from glucose monitoring, many systems have been introduced mainly for application in the central nervous system in animal models. We compare the microsensor principle with other methods applied in biomedical research to show advantages and drawbacks. Electrochemical sensor systems are easily miniaturized and fabricated by microtechnology processes. We review different microfabrication approaches for in vivo sensor platforms, ranging from simple modified wires and fibres to fully microfabricated systems on silicon, ceramic or polymer substrates. The various immobilization methods for the enzyme such as chemical cross-linking and entrapment in polymer membranes are discussed. The resulting sensor performance is compared in detail. We also examine different concepts to reject interfering substances by additional membranes, aspects of instrumentation and biocompatibility. Practical considerations are elaborated, and conclusions for future developments are presented. Graphical Abstract ᅟ
Keywords: In vivo; Biosensor; Chemical sensor; Microfabrication; Glutamate; Lactate

Disc-based microarrays: principles and analytical applications by Sergi Morais; Rosa Puchades; Ángel Maquieira (4523-4534).
The idea of using disk drives to monitor molecular biorecognition events on regular optical discs has received considerable attention during the last decade. CDs, DVDs, Blu-ray discs and other new optical discs are universal and versatile supports with the potential for development of protein and DNA microarrays. Besides, standard disk drives incorporated in personal computers can be used as compact and affordable optical reading devices. Consequently, a CD technology, resulting from the audio–video industry, has been used to develop analytical applications in health care, environmental monitoring, food safety and quality assurance. The review presents and critically evaluates the current state of the art of disc-based microarrays with illustrative examples, including past, current and future developments. Special mention is made of the analytical developments that use either chemically activated or raw standard CDs where proteins, oligonucleotides, peptides, haptens or other biological probes are immobilized. The discs are also used to perform the assays and must maintain their readability with standard optical drives. The concept and principle of evolving disc-based microarrays and the evolution of disk drives as optical detectors are also described. The review concludes with the most relevant uses ordered chronologically to provide an overview of the progress of CD technology applications in the life sciences. Also, it provides a selection of important references to the current literature. Graphical Abstract High density disc-based microarrays
Keywords: Consumer electronics; Compact disc; Biosensing; Molecular recognition; Point of care

Raman-based microarray readout: a review by Christoph Haisch (4535-4545).
For a quarter of a century, microarrays have been part of the routine analytical toolbox. Label-based fluorescence detection is still the commonest optical readout strategy. Since the 1990s, a continuously increasing number of label-based as well as label-free experiments on Raman-based microarray readout concepts have been reported. This review summarizes the possible concepts and methods and their advantages and challenges. A common label-based strategy is based on the binding of selective receptors as well as Raman reporter molecules to plasmonic nanoparticles in a sandwich immunoassay, which results in surface-enhanced Raman scattering signals of the reporter molecule. Alternatively, capture of the analytes can be performed by receptors on a microarray surface. Addition of plasmonic nanoparticles again leads to a surface-enhanced Raman scattering signal, not of a label but directly of the analyte. This approach is mostly proposed for bacteria and cell detection. However, although many promising readout strategies have been discussed in numerous publications, rarely have any of them made the step from proof of concept to a practical application, let alone routine use. Graphical Abstract Possible realization of a SERS (Surface-Enhanced Raman Scattering) system for microarray readout
Keywords: Raman spectroscopy; Surface-enhanced Raman spectroscopy; Surface-enhanced resonance Raman scattering; Microarray; Readout

Determination of elemental impurities in pharmaceutical products and related matrices by ICP-based methods: a review by Juliano S. Barin; Paola A. Mello; Marcia F. Mesko; Fabio A. Duarte; Erico M. M. Flores (4547-4566).
Interest in the determination of elemental impurities in pharmaceuticals has increased in recent years because of changes in regulatory requirements and the need for changing or updating the current limit tests recommended in pharmacopeias. Inductively coupled plasma (ICP) optical emission spectrometry and ICP mass spectrometry are suitable alternatives to perform multielemental analysis for this purpose. The main advantages and limitations of these techniques are described, covering the applications reported in the literature in the last 10 years mainly for active pharmaceutical ingredients, raw materials, and pharmaceutical dosage forms. Strategies used for sample preparation, including dissolution in aqueous or organic solvents, extraction, wet digestion and combustion methods are described, as well as direct solid analysis and ICP-based systems applied for speciation analysis. Interferences observed during the analysis of pharmaceutical products using ICP-based methods are discussed. Methods currently recommended by pharmacopeias for elemental impurities are also covered, showing that the use of ICP-based methods could be considered as a trend in the determination of these impurities in pharmaceuticals. However, the development of a general method that is accurate for all elemental impurities and the establishment of an official method are still challenges. In this regard, the main drawbacks and suitable alternatives are discussed.
Keywords: Determination of elemental impurities; Pharmacopoeia; Atomic spectrometry; Sample preparation; Heavy metals; Pharmaceutical analysis

Post-SELEX optimization of aptamers by Shunxiang Gao; Xin Zheng; Binghua Jiao; Lianghua Wang (4567-4573).
Aptamers are functional single-stranded DNA or RNA oligonucleotides, selected in vitro by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which can fold into stable unique three-dimensional structures that bind their target ligands with high affinity and specificity. Although aptamers show a number of favorable advantages such as better stability and easier modification when compared with the properties of antibodies, only a handful of aptamers have entered clinical trials and only one, pegaptanib, has received US Food and Drug Administration approval for clinical use. The main reasons that limit the practical application of aptamers are insufficient nuclease stability, bioavailability, thermal stability, or even affinity. Some aptamers obtained from modified libraries show better properties; however, polymerase amplification of nucleic acids containing non-natural bases is currently a primary drawback of the SELEX process. This review focuses on several post-SELEX optimization strategies of aptamers identified in recent years. We describe four common methods in detail: truncation, chemical modification, bivalent or multivalent aptamer construction, and mutagenesis. We believe that these optimization strategies should improve one or more specific properties of aptamers, and the type of feature(s) selected for improvement will be dependent on the application purpose.
Keywords: Aptamer; Optimization; Truncation; Chemical modification; Polyvalent ligand; Mutation

DNA enrichment approaches to identify unauthorized genetically modified organisms (GMOs) by Alfred J. Arulandhu; Jeroen P. van Dijk; David Dobnik; Arne Holst-Jensen; Jianxin Shi; Jana Zel; Esther J. Kok (4575-4593).
With the increased global production of different genetically modified (GM) plant varieties, chances increase that unauthorized GM organisms (UGMOs) may enter the food chain. At the same time, the detection of UGMOs is a challenging task because of the limited sequence information that will generally be available. PCR-based methods are available to detect and quantify known UGMOs in specific cases. If this approach is not feasible, DNA enrichment of the unknown adjacent sequences of known GMO elements is one way to detect the presence of UGMOs in a food or feed product. These enrichment approaches are also known as chromosome walking or gene walking (GW). In recent years, enrichment approaches have been coupled with next generation sequencing (NGS) analysis and implemented in, amongst others, the medical and microbiological fields. The present review will provide an overview of these approaches and an evaluation of their applicability in the identification of UGMOs in complex food or feed samples.
Keywords: GMOs; UGMOs; PCR; NGS; Enrichment approaches

Application of whole genome shotgun sequencing for detection and characterization of genetically modified organisms and derived products by Arne Holst-Jensen; Bjørn Spilsberg; Alfred J. Arulandhu; Esther Kok; Jianxin Shi; Jana Zel (4595-4614).
The emergence of high-throughput, massive or next-generation sequencing technologies has created a completely new foundation for molecular analyses. Various selective enrichment processes are commonly applied to facilitate detection of predefined (known) targets. Such approaches, however, inevitably introduce a bias and are prone to miss unknown targets. Here we review the application of high-throughput sequencing technologies and the preparation of fit-for-purpose whole genome shotgun sequencing libraries for the detection and characterization of genetically modified and derived products. The potential impact of these new sequencing technologies for the characterization, breeding selection, risk assessment, and traceability of genetically modified organisms and genetically modified products is yet to be fully acknowledged. The published literature is reviewed, and the prospects for future developments and use of the new sequencing technologies for these purposes are discussed.
Keywords: Cisgene; Intragene; Traceability; Transcriptome sequencing; Transgene; Unknown GMO

Advances in DNA metabarcoding for food and wildlife forensic species identification by Martijn Staats; Alfred J. Arulandhu; Barbara Gravendeel; Arne Holst-Jensen; Ingrid Scholtens; Tamara Peelen; Theo W. Prins; Esther Kok (4615-4630).
Species identification using DNA barcodes has been widely adopted by forensic scientists as an effective molecular tool for tracking adulterations in food and for analysing samples from alleged wildlife crime incidents. DNA barcoding is an approach that involves sequencing of short DNA sequences from standardized regions and comparison to a reference database as a molecular diagnostic tool in species identification. In recent years, remarkable progress has been made towards developing DNA metabarcoding strategies, which involves next-generation sequencing of DNA barcodes for the simultaneous detection of multiple species in complex samples. Metabarcoding strategies can be used in processed materials containing highly degraded DNA e.g. for the identification of endangered and hazardous species in traditional medicine. This review aims to provide insight into advances of plant and animal DNA barcoding and highlights current practices and recent developments for DNA metabarcoding of food and wildlife forensic samples from a practical point of view. Special emphasis is placed on new developments for identifying species listed in the Convention on International Trade of Endangered Species (CITES) appendices for which reliable methods for species identification may signal and/or prevent illegal trade. Current technological developments and challenges of DNA metabarcoding for forensic scientists will be assessed in the light of stakeholders’ needs.
Keywords: Endangered species; Next-generation sequencing; Wildlife forensic samples; Cytochrome c oxidase I; Convention on International Trade of Endangered Species

The biochemical origins of the surface-enhanced Raman spectra of bacteria: a metabolomics profiling by SERS by W. Ranjith Premasiri; Jean C. Lee; Alexis Sauer-Budge; Roger Théberge; Catherine E. Costello; Lawrence D. Ziegler (4631-4647).
is a research scientist (chemistry) at the Photonics Center at Boston University. He is an analytical chemist with expertise in vibrational and surface-enhanced Raman spectroscopy (SERS). He is the inventor of a SERS substrate and portable Raman spectrometer. He is interested in applications of SERS in trace analyses and diagnostics. is Associate Professor of Medicine (infectious diseases) at the Brigham and Women’s Hospital and Harvard Medical School. She is a medical microbiologist with specific expertise in the pathogenesis of Staphylococcus aureus infections. She has special interests in the staphylococcal capsular polysaccharides, their biosynthesis, function, and regulation, and S. aureus vaccine development. is Head of the Biomedical Group at Fraunhofer Center for Manufacturing Innovation and Adjunct Research Assistant Professor in the Biomedical Engineering Department at Boston University. Her research interests include the development of novel infectious disease diagnostics, tissue engineering, biomedical devices, and scientific instrumentation. is a senior research scientist at the Center for Biomedical Mass Spectrometry at the Boston University School of Medicine. He has been working on potential uses of top-down mass spectrometry for the detection and characterization of hemoglobinopathies, and this approach has been adapted to a variety of platforms to evaluate potential clinical applications. His main area of interest is the application of mass spectrometry in the clinical laboratory. is a William Fairfield Warren Distinguished Professor at Boston University, holding appointments in biochemistry, biophysics and chemistry, and is President of the International Mass Spectrometry Foundation. Her research centers on development and application of mass-spectrometry-based methods to study protein posttranslational modifications and folding disorders, cardiovascular and infectious diseases, Glycobiology, and bioactive lipids. is Professor and Chair of the Department of Chemistry at Boston University and a member of the Boston University Photonics Center. He is also Senior Associate Editor of the Journal of Raman Spectroscopy. He has developed a number of novel molecular frequency-domain and time-domain spectroscopic techniques. His current research interests include ultrafast spectroscopic studies of short time dynamics of chemical and materials systems and the development of surface-enhanced Raman spectroscopy for a variety of bioanalytical applications. The dominant molecular species contributing to the surface-enhanced Raman spectroscopy (SERS) spectra of bacteria excited at 785 nm are the metabolites of purine degradation: adenine, hypoxanthine, xanthine, guanine, uric acid, and adenosine monophosphate. These molecules result from the starvation response of the bacterial cells in pure water washes following enrichment from nutrient-rich environments. Vibrational shifts due to isotopic labeling, bacterial SERS spectral fitting, SERS and mass spectrometry analysis of bacterial supernatant, SERS spectra of defined bacterial mutants, and the enzymatic substrate dependence of SERS spectra are used to identify these molecular components. The absence or presence of different degradation/salvage enzymes in the known purine metabolism pathways of these organisms plays a central role in determining the bacterial specificity of these purine-base SERS signatures. These results provide the biochemical basis for the development of SERS as a rapid bacterial diagnostic and illustrate how SERS can be applied more generally for metabolic profiling as a probe of cellular activity. Graphical Abstract Bacterial typing by metabolites released under stress
Keywords: Surface-enhanced Raman spectroscopy; Bacteria; Nucleotide degradation; Purine metabolism; Metabolic profiling

Concurrent supercritical fluid chromatographic analysis of terpene lactones and ginkgolic acids in Ginkgo biloba extracts and dietary supplements by Mei Wang; Emily J. Carrell; Amar G. Chittiboyina; Bharathi Avula; Yan-Hong Wang; Jianping Zhao; Jon F. Parcher; Ikhlas A. Khan (4649-4660).
Supercritical fluid chromatography was used to resolve and determine ginkgolic acids (GAs) and terpene lactones concurrently in ginkgo plant materials and commercial dietary supplements. Analysis of GAs (C13:0, C15:0, C15:1, and C17:1) was carried out by ESI (−) mass detection. The ESI (−) spectra of GAs simply displayed only the [M-H] pseudo-molecular ions, and selected ion monitoring (SIM) for those ions was used for the quantification. Analysis of terpene lactones (ginkgolides A, B, C, J and bilobalide) was complicated by in-source collision-induced dissociation (IS-CID) in the ESI source. Thus, MS analysis could be influenced by the fragmentation pattern produced by the IS-CID. However, it was established that the fragmentation pattern, measured by ion survival yield (ISY), was independent of analyte concentration or matrix at a fixed cone voltage in the ESI source. Therefore, MS with SIM mode was applicable for the analysis of these analytes. The reported method provided consistent and sensitive analysis for the analytes of interest. The LOQs and LODs were determined to be below 100 and 40 ng/mL for GAs and 1 μg/mL and 400 ng/mL for terpene lactones, respectively. Intra- and inter-day precisions were found to be satisfactory with RSDs being below 5.2 %. Analyte recoveries ranged from 87 to 109 %. The developed method was successfully applied to the analysis of 11 ginkgo plant samples and 8 dietary supplements with an analysis time of less than 12 min.
Keywords: Ginkgo biloba ; Terpene lactones; Ginkgolic acids; Supercritical fluid chromatography/photo diode array-mass spectrometry (SFC/PDA-MS); In-source collision-induced dissociation (IS-CID); Dietary supplements

A GC/MS method for determination of succinylacetone in Arabidopsis thaliana by Lizi Zhou; Zhaoguang Yang; Tiantian Zhi; Zhou Zhou; Xiaochen Wang; Chunmei Ren; Bo Qiu (4661-4667).
Succinylacetone was known to be a toxic metabolite of tyrosine in human and animals caused by blockage of the final step in tyrosine degradation pathway, but its existence in plant was unclear though the metabolic disturbance of tyrosine was also found in plant. A GC-MS method for determination of succinylacetone in Arabidopsis thaliana was developed for the first time. Both oximation and silylation were applied in the derivation procedure, and a low-temperature condition before completion of oximation was found to be necessary to obtain good linearity of the calibration curve due to the thermolability of succinylacetone. The specific chromatogram pattern formed by the four isomers of succinylacetone derivatives provided a helpful feature for its identification. The detection limit of the proposed method was 0.25 ppm in A. thaliana. The recoveries were between 95.4 and 109.3 % with the coefficient of variation ranging from 4.36 to 7.81 % for intra-day assays and 6.47 to 8.52 % for inter-day assays. Application to wild-type and the short-day sensitive cell death 1 mutant of A. thaliana represented an obvious correlation between the measured amount of succinylacetone and wilting symptom, suggesting the proposed method could be a powerful tool in further study on toxicology of succinylacetone and tyrosine catabolism in plants.
Keywords: Succinylacetone; Arabidopsis thaliana ; Tyrosine catabolism; Gas chromatography; Mass spectrometry

Identification and absolute quantification of enzymes in laundry detergents by liquid chromatography tandem mass spectrometry by Alexandra Gaubert; Jérémy Jeudy; Blandine Rougemont; Claire Bordes; Jérôme Lemoine; Hervé Casabianca; Arnaud Salvador (4669-4681).
In a stricter legislative context, greener detergent formulations are developed. In this way, synthetic surfactants are frequently replaced by bio-sourced surfactants and/or used at lower concentrations in combination with enzymes. In this paper, a LC-MS/MS method was developed for the identification and quantification of enzymes in laundry detergents. Prior to the LC-MS/MS analyses, a specific sample preparation protocol was developed due to matrix complexity (high surfactant percentages). Then for each enzyme family mainly used in detergent formulations (protease, amylase, cellulase, and lipase), specific peptides were identified on a high resolution platform. A LC-MS/MS method was then developed in selected reaction monitoring (SRM) MS mode for the light and corresponding heavy peptides. The method was linear on the peptide concentration ranges 25–1000 ng/mL for protease, lipase, and cellulase; 50–1000 ng/mL for amylase; and 5–1000 ng/mL for cellulase in both water and laundry detergent matrices. The application of the developed analytical strategy to real commercial laundry detergents enabled enzyme identification and absolute quantification. For the first time, identification and absolute quantification of enzymes in laundry detergent was realized by LC-MS/MS in a single run. Graphical Abstract Identification and quantification of enzymes by LC-MS/MS
Keywords: Protein; Enzyme; Laundry detergent; Mass spectrometry; Sample preparation

Metabolite profiling of urine has seen much advancement in recent years, and its analysis by nuclear magnetic resonance (NMR) spectroscopy has become well established. However, the highly variable nature of human urine still requires improved protocols despite some standardization. In particular, diseases such as kidney disease can have a profound effect on the composition of urine and generate a highly diverse sample set for clinical studies. Large variations in pH and the cationic concentration of urine play an important role in creating positional noise within datasets generated from NMR. We demonstrate positional noise to be a confounding variable for multivariate statistical tools such as statistical total correlation spectroscopy (STOCSY), thereby hindering the process of biomarker discovery. We present a two-dimensional buffering system using potassium fluoride (KF) and phosphate buffer to reduce positional noise in metabolomic data generated from urine samples with various levels of proteinuria. KF reduces positional noise in citrate peaks, by decreasing the mean relative standard deviation (RSD) from 0.17 to 0.09. By reducing positional noise with KF, STOCSY analysis of citrate peaks saw significant improvement. We further aligned spectral data using a recursive segment-wise peak alignment (RSPA) method, which leads to further improvement of the positional noise (RSD = 0.06). These results were validated using diverse selection of metabolites which lead to an overall improvement in positional noise using the suggested protocol. In summary, we provide an improved workflow for urine metabolite biomarker discovery to achieve higher data quality for better pathophysiological understanding of human diseases. Graphical abstract Citrate peaks in the range 2.75–2.5 ppm from datasets with different sample preparation protocols and with/without in silico alignment. A Citrate peaks with standard phosphate buffering and without in silico alignment. B citrate peaks with standard phosphate buffering and with in silico alignment. C citrate peak with additional potassium fluoride and standard phosphate buffering without in silico alignment. D citrate peaks with additional potassium fluoride and standard phosphate buffering with in silico alignment. Below the respective spectrum are displayed the percent relative standard deviation (RSD) of the respective citrate peaks. This is a measure of the positional noise of peaks within a 1H NMR analysis. It can be seen that D performs the best in reducing positional noise of citrate peaks. EH STOCSY analysis of correlating spectral features with the driver peak at 2.675 ppm (see red arrow) to identify structural correlations. As a, b, c, and d are known to be structurally correlated, STOCSY analysis should reveal r 2 = 1 if data is perfectly aligned and can therefore be used as a measure of peak alignment. E Strong positional noise does not allow identifying the c and d peaks of the AB system to be correlated. F, G Neither in silico alignment or KF addition alone can completely improve the alignment and therefore increase the correlations. H Highly improved alignment by combining both KF addition and in silico alignment reduces positional noise and elucidates all four citrate peaks to be strongly correlated
Keywords: Metabolomics; Non-targeted; Multivariate data analysis; Biomarker discovery; Urine; Nuclear magnetic resonance spectroscopy

The red imported fire ant, Solenopsis invicta, is an aggressive, highly invasive pest ant species from South America that has been introduced into North America, Asia, and Australia. Quarantine efforts have been imposed in the USA to minimize further spread of the ant. To aid the quarantine efforts, there remains an acute need for a rapid, field portable method for the identification of these ants. In this report, we describe two novel monoclonal antibodies that specifically bind the S. invicta venom protein 2 produced by S. invicta. Using these monoclonal antibodies we developed a lateral flow immunoassay that provides a rapid and portable method for the identification of S. invicta ants. The lateral flow immunoassay was validated against purified S. invicta venom protein 2 and 33 unique ant species (representing 15 % of the total species and 42 % of the Myrmicinae genera found in Florida), and only S. invicta and the S. invicta/richteri hybrid produced a positive result. These monoclonal antibodies were selective to S. invicta venom protein 2 and did not bind to proteins from congeners (i.e., S. geminata or S. richteri) known to produce a S. invicta venom protein 2 ortholog. This S. invicta lateral flow immunoassay provides a new tool for regulatory agencies in the USA to enforce quarantine protocols and limit the spread of this invasive ant. Graphical Abstract Field method to detect and identify the red imported fire ant, Solenopsis invicta
Keywords: Solenopsis invicta ; Solenopsis richteri ; Fire ant; Venom; Antibodies; Lateral flow immunoassay

A novel enzyme-free hydrogen peroxide sensor composed of carbon dots (CDs) and multi-walled carbon nanotubes (MWCNTs) was prepared. It was found that the carbon dots-decorated multi-walled carbon nanotubes nanocomposites (CDs/MWCNTs) modified glassy carbon (GC) electrode (CDs/MWCNTs/GCE) exhibited a significant synergistic electrocatalytic activity towards hydrogen peroxide reduction as compared to carbon dots or multi-walled carbon nanotubes alone, and the CDs/MWCNTs/GCE has shown a low detection limit as well as excellent stability, selectivity, and reproducibility. These remarkable analytical advantages enable the practical application of CDs/MWCNTs/GCE for the real-time tracking of hydrogen peroxide (H2O2) released from human cervical cancer cells with satisfactory results. The enhanced electrochemical activity can be assigned to the edge plane-like defective sites and lattice oxygen in the CDs/MWCNTs nanocomposites due to the small amount of decoration of carbon dots on the multi-walled carbon nanotubes. Based on a facile preparation method and with good electrochemical properties, the CDs/MWCNTs nanocomposites represent a new class of carbon electrode for electrochemical sensor applications. Graphical Abstract CDs/MWCNTs exhibited good electrocatalytic activity and stability to H2O2 reduction and can be used for real-time detection of H2O2 released from living cells.
Keywords: Carbon quantum dots; Catalyst; Extracellular H2O2 ; Electrocatalysis; Nonenzymatic biosensor

We describe a sensitive aptamer-based sandwich assay for protein detection on microplate by using rolling circle amplification (RCA) coupled with thrombin catalysis. This assay takes advantage of RCA generating long DNA oligonucleotides with repeat thrombin-binding aptamer sequence, specific aptamer affinity binding to achieve multiple thrombin labeling, and enzyme activity of thrombin for signal generation. Protein target is specifically captured by antibody-coated microplate. Then, an oligonucleotide containing an aptamer for protein and a primer sequence is added to form a typical sandwich structure. Following a template encoded with complementary sequence of aptamer for thrombin, RCA reaction extends the primer sequence into a long oligonucleotide. Many thrombin molecules bind with the RCA product. Thrombin catalyzes the conversion of its chromogenic or fluorogenic peptide substrates into detectable products for final quantification of protein targets. We applied this strategy to the detection of a model protein target, platelet-derived growth factor-BB (PDGF-BB). Due to double signal amplifications from RCA and thrombin catalysis, this assay enabled the detection of PDGF-BB as low as 3.1 pM when a fluorogenic peptide substrate was used. This assay provides a new way for signal generation in RCA-involved assay through direct thrombin labeling, circumventing time-consuming preparation of enzyme-conjugate and affinity probes. This method has promise for a variety of analytical applications.
Keywords: Aptamer; Thrombin; Rolling circle amplification; Multiple enzyme labeling; Signal amplification; Protein detection

Vitamin C (ascorbic acid, AA) is very labile in nature and decays quickly after blood withdrawal. To ensure AA stability, the current procedure prescribes immediate plasma acidification followed by sample storage at ultra-low temperature. The aim of this study was to set up a pre-analytical procedure to promptly stabilize AA at routine temperatures while minimizing both specimen manipulation and instrumental requirement. Blood from healthy subjects was collected in lithium-heparin gel separator tubes containing or not different reducing agents (dithioerythritol, tris(2-carboxyethyl)phosphine, n-acetylcysteine and sodium thiosulfate). Plasma AA stability during blood and plasma storage at routine temperatures was evaluated. Plasma AA concentration was assayed by RP-HPLC-UV under ion suppression conditions. Each of the reductants tested was able to slow down the ex vivo degradation of plasma AA; dithioerythritol was the most effective. Five to 10 mmol/L dithioerythritol did not interfere with blood separation and allowed plasma AA to be stabilized up to 6 h, 24 h and 60 days at room temperature, +4 °C and −25 °C, respectively. The method worked well even in case of delayed blood separation and/or incomplete vacutainer filling. The procedure is feasible and reliable. Of special usefulness in clinical and epidemiological studies, prompt plasma manipulation after blood withdrawal or special storage equipments are not required. Graphical Abstract Collecting blood in tubes containing a reducing agent is a feasible method to promptly and effectively stabilize plasma vitamin C at routine temperature
Keywords: Ascorbic acid; Blood specimen collection; Specimen handling; Antioxidants; Dithioerythritol; Reverse-phase chromatography

In this contribution, the utility of sequential injection analysis in combination with surface-enhanced Raman spectroscopy (SERS) as a detection technique was investigated for simultaneous determination of aspirin and vitamin C in their pharmaceutical dosage forms and in spiked urine samples. The silver substrate was synthesized in situ by laser-induced photochemical procedure. By focusing the laser on a flow cell at 1 ml/min of continuous flow of 0.5 mM silver nitrate and 5 mM sodium citrate mixture, an active silver spot on the inner wall of the flow cell was prepared in a few seconds. The whole setup is fully computer controlled using ATLAS software to combine the two techniques. The system allows sequential determination of aspirin concentrations ranging from 100 to 500 ng/ml and vitamin C concentrations between 10 and 110 ng/ml with good precision of relative standard deviations (RSDs) of 0.85 and 1.7 %, respectively. A comparison of these results with those of the reported procedures showed excellent results compared with t and F values, indicating good accuracy and precision. The detection limits were 32 and 3 ng/ml for aspirin and vitamin C, respectively.
Keywords: SERS; Sequential injection analysis; Aspirin; Vitamin C; In situ silver photoreduction

Optimization and comparison of bottom-up proteomic sample preparation for early-stage Xenopus laevis embryos by Elizabeth H. Peuchen; Liangliang Sun; Norman J. Dovichi (4743-4749).
Xenopus laevis is an important model organism in developmental biology. While there is a large literature on changes in the organism’s transcriptome during development, the study of its proteome is at an embryonic state. Several papers have been published recently that characterize the proteome of X. laevis eggs and early-stage embryos; however, proteomic sample preparation optimizations have not been reported. Sample preparation is challenging because a large fraction (~90 % by weight) of the egg or early-stage embryo is yolk. We compared three common protein extraction buffer systems, mammalian Cell-PE LBTM lysing buffer (NP40), sodium dodecyl sulfate (SDS), and 8 M urea, in terms of protein extraction efficiency and protein identifications. SDS extracts contained the highest concentration of proteins, but this extract was dominated by a high concentration of yolk proteins. In contrast, NP40 extracts contained ~30 % of the protein concentration as SDS extracts, but excelled in discriminating against yolk proteins, which resulted in more protein and peptide identifications. We then compared digestion methods using both SDS and NP40 extraction methods with one-dimensional reverse-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS). NP40 coupled to a filter-aided sample preparation (FASP) procedure produced nearly twice the number of protein and peptide identifications compared to alternatives. When NP40-FASP samples were subjected to two-dimensional RPLC-ESI-MS/MS, a total of 5171 proteins and 38,885 peptides were identified from a single stage of embryos (stage 2), increasing the number of protein identifications by 23 % in comparison to other traditional protein extraction methods.
Keywords: Developmental biology; Embryology; Mass spectrometry; Proteomics; Xenopus laevis

Multi-platform metabolomics assays for human lung lavage fluids in an air pollution exposure study by Izabella Surowiec; Masoumeh Karimpour; Sandra Gouveia-Figueira; Junfang Wu; Jon Unosson; Jenny A. Bosson; Anders Blomberg; Jamshid Pourazar; Thomas Sandström; Annelie F. Behndig; Johan Trygg; Malin L. Nording (4751-4764).
Metabolomics protocols are used to comprehensively characterize the metabolite content of biological samples by exploiting cutting-edge analytical platforms, such as gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) assays, as well as nuclear magnetic resonance (NMR) assays. We have developed novel sample preparation procedures combined with GC-MS, LC-MS, and NMR metabolomics profiling for analyzing bronchial wash (BW) and bronchoalveolar lavage (BAL) fluid from 15 healthy volunteers following exposure to biodiesel exhaust and filtered air. Our aim was to investigate the responsiveness of metabolite profiles in the human lung to air pollution exposure derived from combustion of biofuels, such as rapeseed methyl ester biodiesel, which are increasingly being promoted as alternatives to conventional fossil fuels. Our multi-platform approach enabled us to detect the greatest number of unique metabolites yet reported in BW and BAL fluid (82 in total). All of the metabolomics assays indicated that the metabolite profiles of the BW and BAL fluids differed appreciably, with 46 metabolites showing significantly different levels in the corresponding lung compartments. Furthermore, the GC-MS assay revealed an effect of biodiesel exhaust exposure on the levels of 1-monostearylglycerol, sucrose, inosine, nonanoic acid, and ethanolamine (in BAL) and pentadecanoic acid (in BW), whereas the LC-MS assay indicated a shift in the levels of niacinamide (in BAL). The NMR assay only identified lactic acid (in BW) as being responsive to biodiesel exhaust exposure. Our findings demonstrate that the proposed multi-platform approach is useful for wide metabolomics screening of BW and BAL fluids and can facilitate elucidation of metabolites responsive to biodiesel exhaust exposure. Graphical Abstract Graphical abstract illustrating the study workflow. NMR Nuclear Magnetic Resonance, LC-TOFMS Liquid chromatography-Time Of Flight Mass Spectrometry, GC Gas Chromatography-Mass spectrometry
Keywords: GC-MS; LC-MS; NMR; Biodiesel; Bronchoalveolar lavage

N-Glycan profile analysis of transferrin using a microfluidic compact disc and MALDI-MS by Alessandro Quaranta; Anna Sroka-Bartnicka; Erik Tengstrand; Gunnar Thorsén (4765-4776).
It has been known for a long time that diseases can be associated with changes to the glycosylation of specific proteins. This has been shown for cancer, immunological disorders, and neurodegenerative diseases. The possibility of using the glycosylation patterns of proteins as biomarkers for disease would be a great asset for clinical research or diagnosis. There is at present a lack of rapid, automated, and cost-efficient analytical techniques for the determination of the glycosylation of specific serum proteins. We have developed a method for determining the glycosylation pattern of proteins based on the affinity capture of a specific serum protein, the enzymatic release of the N-linked glycans, and the analysis of the glycan pattern using MALDI-MS. All sample preparation is performed in a disposable centrifugal microfluidic disc. The sample preparation is miniaturized, requiring only 1 μL of sample per determination, and automated with the possibility of processing 54 samples in parallel in 3.5 h. We have developed a method for the glycosylation pattern analysis of transferrin. The method has been tested on serum samples from chronic alcohol abusers and a control group. Also, a SIMCA model was created and evaluated to discriminate between the two groups.
Keywords: Glycan pattern; Glycomics; N-linked oligosaccharide; Transferrin; Microfluidic; MALDI-MS

The use of metabonomic methodologies to identify illicit salbutamol administration in cattle has not been previously investigated. In this study, a nuclear magnetic resonance (NMR)-based untargeted quantitative metabonomic approach was applied to discriminate biofluid samples (plasma and urine) obtained from cattle before and after salbutamol treatment. Six male cattle (265.7 ± 3.9 kg) were fed salbutamol (0.15 mg/kg body weight) for 21 consecutive days. Plasma and urine samples were collected before and after treatment. By the use of targeted profiling, 46 and 43 metabolites in plasma and urine, respectively, were quantified, of which 9 and 11 metabolites were significantly affected (P < 0.05) by salbutamol treatment. Partial least squares discriminant analysis showed that both plasma and urine samples collected after treatment were well separated from those before treatment, with Q 2 values of 0.56 and 0.573 for plasma and urine samples, respectively. The variable importance plot (VIP) scores of glucose and lactate in plasma, and urine, hippurate, acetate, glycine, formate, n-phenylacetyl, benzoate, and phenylacetate in urine were >1.0, which implies that these metabolites may serve as potential biomarkers for salbutamol treatment. These findings suggest the potential value of NMR-based untargeted quantitative metabonomic methodologies for plasma and urine analyses as a screening technique for detection of illicit salbutamol usage in cattle.
Keywords: NMR; Quantitative metabonomics; Salbutamol; Plasma; Urine; Cattle

Detection of tumor-related proteins with high specificity and sensitivity is important for early diagnosis and prognosis of cancers. While protein sensors based on antibodies are not easy to keep for a long time, aptamers (single-stranded DNA) are found to be a good alternative for recognizing tumor-related protein specifically. This study investigates the feasibility of employing aptamers to recognize the platelet-derived growth factor (PDGF) specifically and subsequently triggering rolling circle amplification (RCA) of DNAs on extended-gate field-effect transistors (EGFETs) to enhance the sensitivity. The EGFETs are fabricated by the standard CMOS technology and integrated with readout circuits monolithically. The monolithic integration not only avoids the wiring complexity for a large sensor array but also enhances the sensor reliability and facilitates massive production for commercialization. With the RCA primers immobilized on the sensory surface, the protein signal is amplified as the elongation of DNA, allowing the EGFET to achieve a sensitivity of 8.8 pM, more than three orders better than that achieved by conventional EGFETs. Moreover, the responses of EGFETs are able to indicate quantitatively the reaction rates of RCA, facilitating the estimation on the protein concentration. Our experimental results demonstrate that immobilized RCA on EGFETs is a useful, label-free method for early diagnosis of diseases related to low-concentrated tumor makers (e.g., PDGF) for serum sample, as well as for monitoring the synthesis of various DNA nanostructures in real time. Graphical Abstract The tumor-related protein, PDGF, is detected by immobilizing rolling circle amplification on an EGFET with integrated readout circuit
Keywords: Real-time monitoring; Label-free detection; Rolling circle; Amplification; Extended-gate field-effect transistors; Monolithically integrated circuits

Plasma amine oxidase (PAO), which is also designated as semicarbazide-sensitive amine oxidase (SSAO), copper-containing amine oxidase 3 (AOC3), or vascular adhesion protein-1 (VAP-1), catalyzes the oxidative deamination of primary amines to aldehydes using copper and a quinone as cofactors. Because it participates in the transmigration of inflammatory cells through the blood vessels into the tissue, PAO is attributed an important role in inflammatory diseases. Therefore, inhibitors of this enzyme could lead to new therapeutics for the treatment of inflammation-related conditions. Assays for the evaluation of PAO inhibitors usually measure the conversion of benzylamine to benzaldehyde by UV spectroscopy. We have developed a test system with the new substrate 6-(5-phenyl-2H-tetrazol-2-yl)hexan-1-amine, monitoring the formation of the enzyme product 6-(5-phenyl-2H-tetrazol-2-yl)hexanal by reversed phase HPLC with UV detection. Since this compound only eluted with poor peak shape due to hydrate formation in the aqueous mobile phase, it was derivatized with tris(hydroxymethyl)aminomethane (TRIS) under mild conditions to an oxazolidine prior HPLC analysis. The validation of the method revealed that the new substrate was bound with higher affinity to PAO and converted with higher velocity than the standard substrate benzylamine.
Keywords: Plasma amine oxidase; Vascular adhesion protein-1; Inhibitor screening; Aliphatic aldehyde; Derivatization; TRIS

Noting the source-dependent properties of dissolved organic matter (DOM), this study explored the recoverable compounds by solid phase extraction (SPE) of two common sorbents (C18 and PPL) eluted with methanol solvent for contrasting DOM sources via fluorescence excitation-emission matrix coupled with parallel factor analysis (EEM-PARAFAC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Fresh algae and leaf litter extracts DOM, one riverine DOM, and one upstream lacustrine DOM were selected for the comparison. C18 sorbent was generally found to extract more diverse molecular formula, relatively higher molecular weight, and more heteroatomic DOM compounds within the studied mass range than PPL sorbent except for the leaf litter extract. Even with the same sorbent, the main molecular features of the two end member DOM were distributed on different sides of the axes of a multivariate ordination, indicating the source-dependent characteristics of the recoverable compounds by the sorbents. In addition, further examination of the molecular formula uniquely present in the two end members and the upstream lake DOM suggested that proteinaceous, tannin-like, and heteroatomic DOM constituents might be potential compound groups which are labile and easily degraded during their mobilization into downstream watershed. This study provides new insights into the sorbent selectivity of DOM from diverse sources and potential lability of various compound groups.
Keywords: Dissolved organic matter (DOM); FT-ICR-MS; Sorbent selectivity; Solid phase extraction; Lability

Erratum to: LC-MS/MS determination of potential endocrine disruptors of cortico signalling in rivers and wastewaters by Adrian A. Ammann; Petra Macikova; Ksenia J. Groh; Kristin Schirmer; Marc J.-F. Suter (4821-4822).