Analytical and Bioanalytical Chemistry (v.410, #4)

Microbial biosensors for analytical applications by Gérald Thouand (1189-1190).
received his PhD degree in microbiology from the University of Nancy, France, in 1993. He is a tenured professor in microbiology at the University of Nantes. Deputy for Research Innovation at the French Ministry of Research (DRRT Pays de La Loire) and Auditor at the Institute of Higher Studies for Science and Technology (IHEST, Paris), his research interests include environmental monitoring of biodegradation and biotechnology using microbial biosystems. He is mainly involved in the development of biosensors for the detection of chemical pollutants and pathogenic bacteria. He is a member of the Society for Applied Microbiology (SFAM, UK) and a member of the scientific committee of the International Society of Bioluminescence and Chemiluminescence. He is Associate Editor for Frontiers in Microbiology and Editor for Environmental Science and Pollution Research (ESPR, Springer). He has authored 56 international peer-reviewed articles, 4 patents, and 16 book chapters or editions; presented at 60 international conferences; and supervised more than 20 PhD students.

Synthetic biology for microbial heavy metal biosensors by Hyun Ju Kim; Haeyoung Jeong; Sang Jun Lee (1191-1203).
Using recombinant DNA technology, various whole-cell biosensors have been developed for detection of environmental pollutants, including heavy metal ions. Whole-cell biosensors have several advantages: easy and inexpensive cultivation, multiple assays, and no requirement of any special techniques for analysis. In the era of synthetic biology, cutting-edge DNA sequencing and gene synthesis technologies have accelerated the development of cell-based biosensors. Here, we summarize current technological advances in whole-cell heavy metal biosensors, including the synthetic biological components (bioparts), sensing and reporter modules, genetic circuits, and chassis cells. We discuss several opportunities for improvement of synthetic cell-based biosensors. First, new functional modules must be discovered in genome databases, and this knowledge must be used to upgrade specific bioparts through molecular engineering. Second, modules must be assembled into functional biosystems in chassis cells. Third, heterogeneity of individual cells in the microbial population must be eliminated. In the perspectives, the development of whole-cell biosensors is also discussed in the aspects of cultivation methods and synthetic cells.
Keywords: Synthetic biology; Microbial whole-cell biosensor; Heavy metals

Optical microalgal biosensors for aqueous contaminants using organically doped silica as cellular hosts by Nada Ben Ahmed; Sylvie Masse; Guillaume Laurent; Jean-Yves Piquemal; Claude Yéprémian; Roberta Brayner; Thibaud Coradin (1205-1216).
Optical biosensors for the detection of toxic species in aqueous media were developed via the encapsulation of microalgae in sol–gel matrices. In a first step, the effect of cadmium(II), lead(II), and anthracene on the chlorophyll a fluorescence intensity of Anabaena flos-aquae, Chlorella vulgaris, and Euglena gracilis microalgae in suspension was studied. Complementary ATP-metry measurements demonstrated a direct relationship between optical response and pollutant toxicity, in a cell- and dose-dependent manner. In a second step, microalgae were successfully encapsulated in silicate–colloidal silica nanocomposite matrices. However, a complete loss of cell response to pollutant addition was observed, despite the preservation of cell viability. Introduction of a low amount (5 mol%) of amine- or ethyl-bearing silanes in the matrix formulation allowed the recovery of the sensing capacity of the immobilized microalgae, without impacting on the response time (30 s). Porosimetry and 29Si solid-state NMR spectroscopy showed that the organic moieties are fully integrated into the inorganic network, tuning the ability of the target pollutant to diffuse and reach the encapsulated algae. This versatile strategy could be useful for the easy and fast assessment of contamination levels in polluted waters. Graphical Abstract Microalgal biosensors for aqueous contaminants using organically doped silica as cellular hosts
Keywords: Biosensors; Microalgae; Hybrid materials; Contaminated water

Print to detect: a rapid and ultrasensitive phage-based dipstick assay for foodborne pathogens by Hany Anany; Lubov Brovko; Noha K. El Dougdoug; Jennifer Sohar; Heather Fenn; Nada Alasiri; Tarik Jabrane; Patrice Mangin; M. Monsur Ali; Balamurali Kannan; Carlos D. M. Filipe; Mansel W. Griffiths (1217-1230).
Foodborne pathogens are a burden to the economy and a constant threat to public health. The ability to rapidly detect the presence of foodborne pathogens is a vital component of any strategy towards establishing a safe and secure food supply chain. Bacteriophages (phages) are viruses capable of infecting and replicating within bacteria in a strain-specific manner. The ubiquitous and selective nature of phages makes them ideal for the detection and biocontrol of bacteria. Therefore, the objective of this research was to develop and test a phage-based paper dipstick biosensor for the detection of various foodborne pathogens in food matrices. The first step was to identify the best method for immobilizing phages on paper such that their biological activity (infectivity) was preserved. It was found that piezoelectric inkjet printing resulted in lower loss of phage infectivity when compared with other printing methods (namely gravure and blade coating) and that ColorLok paper was ideally suited to create functional sensors. The phage-based bioactive papers developed with use of piezoelectric inkjet printing actively lysed their target bacteria and retained this antibacterial activity for up to 1 week when stored at room temperature and 80% relative humidity. These bioactive paper strips in combination with quantitative real-time PCR were used for quantitative determination of target bacteria in broth and food matrices. A phage dipstick was used to capture and infect Escherichia coli O157:H7, E. coli O45:H2, and Salmonella Newport in spinach, ground beef and chicken homogenates, respectively, and quantitative real-time PCR was used to detect the progeny phages. A detection limit of 10–50 colony-forming units per millilitre was demonstrated with a total assay time of 8 h, which was the duration of a typical work shift in an industrial setting. This detection method is rapid and cost-effective, and may potentially be applied to a broad range of bacterial foodborne pathogens. Graphical abstract ᅟ
Keywords: Bacteriophage; Detection; Bioactive paper; Biosensor

Bioelectrochemical biosensor for water toxicity detection: generation of dual signals for electrochemical assay confirmation by Yuan Yang; Yan-Zhai Wang; Zhen Fang; Yang-Yang Yu; Yang-Chun Yong (1231-1236).
Toxicity assessment of water is of great important to the safety of human health and to social security because of more and more toxic compounds that are spilled into the aquatic environment. Therefore, the development of fast and reliable toxicity assessment methods is of great interest and attracts much attention. In this study, by using the electrochemical activity of Shewanella oneidensis MR-1 cells as the toxicity indicator, 3,5-dichlorophenol (DCP) as the model toxic compound, a new biosensor for water toxicity assessment was developed. Strikingly, the presence of DCP in the water significantly inhibited the maximum current output of the S. oneidensis MR-1 in a three-electrode system and also retarded the current evolution by the cells. Under the optimized conditions, the maximum current output of the biosensor was proportional to the concentration of DCP up to 30 mg/L. The half maximal inhibitory concentration of DCP determined by this biosensor is about 14.5 mg/L. Furthermore, simultaneous monitoring of the retarded time (Δt) for current generation allowed the identification of another biosensor signal in response to DCP which could be employed to verify the electrochemical result by dual confirmation. Thus, the present study has provided a reliable and promising approach for water quality assessment and risk warning of water toxicity.
Keywords: Water toxicity; Biosensor; 3,5-Dichlorophenol; Shewanella ; Dual signal

A novel bioluminescent NanoLuc yeast-estrogen screen biosensor (nanoYES) with a compact wireless camera for effect-based detection of endocrine-disrupting chemicals by Luca Cevenini; Antonia Lopreside; Maria Maddalena Calabretta; Marcello D’Elia; Patrizia Simoni; Elisa Michelini; Aldo Roda (1237-1246).
The presence of chemicals with estrogenic activity in surface, groundwater, and drinking water poses serious concerns for potential threats to human health and aquatic life. At present, no sensitive portable devices are available for the rapid monitoring of such contamination. Here, we propose a cell-based mobile platform that exploits a newly developed bioluminescent yeast-estrogen screen (nanoYES) and a low-cost compact camera as light detector. Saccharomyces cerevisiae cells were genetically engineered with a yeast codon-optimized variant of NanoLuc luciferase (yNLucP) under the regulation of human estrogen receptor α activation. Ready-to-use 3D-printed cartridges with immobilized cells were prepared by optimizing a new procedure that enables to produce alginate slices with good reproducibility. A portable device was obtained exploiting a compact camera and wireless connectivity enabling a rapid and quantitative evaluation (1-h incubation at room temperature) of total estrogenic activity in small sample volumes (50 μL) with a LOD of 0.08 nM for 17β-estradiol. The developed portable analytical platform was applied for the evaluation of water samples spiked with different chemicals known to have estrogen-like activity. Thanks to the high sensitivity of the newly developed yeast biosensor and the possibility to wireless connect the camera with any smartphone model, the developed configuration is more versatile than previously reported smartphone-based devices, and could find application for on-site analysis of endocrine disruptors. Graphical abstract Wireless effect-based detection of endocrine-disrupting chemicals with nanoYES platform
Keywords: Bioluminescence; NanoLuc luciferase; Endocrine disruptors; Effect-based analysis; Estrogenic activity; Yeast-based biosensor

An autonomously bioluminescent Saccharomyces cerevisiae BLYAhS bioreporter was developed in this study for the simple and rapid detection of dioxin-like compounds (DLCs) and aryl hydrocarbon receptor (AhR) agonists. This recombinant yeast reporter was based on a synthetic bacterial luciferase reporter gene cassette (lux) that can produce the luciferase as well as the enzymes capable of self-synthesizing the requisite substrates for bioluminescent production from endogenous cellular metabolites. As a result, bioluminescent signal production is generated continuously and autonomously without cell lysis or exogenous reagent addition. By linking the expression of the autobioluminescent lux reporter cassette to AhR activation via the use of a dioxin-responsive promoter, the S. cerevisiae BLYAhS bioreporter emitted a bioluminescent signal in response to DLC exposure in a dose-responsive manner. The model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), could be detected within 4 h with a half maximal effective concentration (EC50) of ~ 8.1 nM and a lower detection limit of 500 pM. The autobioluminescent response of BLYAhS to other AhR agonists, including 2,3,7,8-tetrachlorodibenzofuran (TCDF), polychlorinated bisphenyl congener 126 (PCB-126) and 169 (PCB-169), 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD), benzo[a]pyrene (BaP), and β-naphthoflavone (bNF), were also characterized in this study. The non-destructive and reagent-free nature of the BLYAhS reporter assay facilitated near-continuous, automated signal acquisition without additional hands-on effort and cost, providing a simple and cost-effective method for rapid DLC detection.
Keywords: Dioxins/furans; Dioxin-like compound; Aryl hydrocarbon receptor; Bioassays; Bioreporter; Autobioluminescence

We present a rapid and simple approach for sensitive detection of antibiotic residues in food samples based on luminescence induction by live bacterial sensor strains integrated into a CCD-based lens-free optical analyzer (LumiSense). Using ciprofloxacin as a model antibiotic, we demonstrate response times of between 20 and 80 min, and detection thresholds of 8 ng/mL for milk, egg white, and chicken essence, and 64 ng/mL for egg yolk. These values are below the minimal allowed values as defined by European Union regulations. Although not intended to replace traditional analytical equipment and regulation-approved methods, LumiSense and similar systems, sample preparation for which involves only simple mixing, dilution, and homogenization, may nevertheless provide a simple means for high-throughput food sample screening. Graphical abstract Detection of bioluminescence from genetically modified bacteria offers a simple and effective way for monitoring an antibiotic, ciprofloxacin, in milk without prior sample preparation
Keywords: Antibiotics; Whole cell biosensor ; Microbial biosensor; Milk; Eggs; Chicken essence

Development of a kelp powder (Thallus laminariae) Standard Reference Material by Lee L. Yu; Joseph F. Browning; Carolyn Q. Burdette; George C. Caceres; Kaitlyn D. Chieh; W. Clay Davis; Brittany L. Kassim; Stephen E. Long; Karen E. Murphy; Rabia Oflaz; Rick L. Paul; Katherine E. Sharpless; Laura J. Wood; James H. Yen; Rolf Zeisler (1265-1278).
is a research chemist in the Chemical Sciences Division of NIST in Gaithersburg. He serves as a clinical program coordinator for the division, and he heads the nonelectrolyte metals review team of the Joint Committee for Traceability in Laboratory Medicine (JCTLM). He is the project leader for SRM 3232. His current research interests are metallomics and nanometrology. earned a B.S. in geology from University of Maryland in 2017, with a focus in geochemistry, and is pursuing a career in metamorphic petrology and tectonics. In his undergraduate, he conducted a senior thesis research project exploring the utility of tourmaline as a mineral indicator for provenance in a geologic setting. is an analytical chemist in the Organic Chemical Measurement Science Group at the National Institute of Standards and Technology. Her research currently focuses on the measurement of vitamins and vitamin metabolites in clinical and dietary intake matrices for the development of reference materials and techniques. is currently an undergraduate student at the University of Maryland, College Park, on track to graduate with a Bachelor’s degree in Biochemistry in 2018. He has been working as a physical science trainee, characterizing the levels of Cd and Pb in Standard Reference Materials within the Inorganic Measurement Science group of the Chemical Sciences Division at the National Institute of Standards and Technology. He will pursue a Ph.D. in Chemistry after the completion of a Bachelor’s degree. was a NIST Summer High School Internship Program (SHIP) intern and then a US Government Pathways Intern when she contributed to the certification measurement of SRM 3232 using ICP-OES. She has since graduated from James Madison University with a B.S. degree in Psychology and a minor in Special Education, Non-Teaching. She is currently working at the Ivymount School as an Applied Behavior Analysis Instructor. works as a research chemist for the National Institute of Standards and Technology (NIST) in Charleston, SC. He has worked in the area of elemental speciation and metallomics for over 15 years. His research interests are centered mainly in the field of proteomics, with special emphasis in the area of high accuracy measurements of metalloproteins and biomarkers. received a BSc honours degree in Chemistry, and a PhD. in Analytical Chemistry from Imperial College, University of London, UK. He is currently a research chemist in the Chemical Sciences Division, National Institute of Standards and Technology, Charleston, SC, an Associate of the Royal College of Science, and a Fellow of the Royal Society of Chemistry. His current research interests center on trace metal analytical chemical metrology. is a research chemist at the National Institute of Standards and Technology and leader of the nanomaterial metrology team in her group. Her research focuses on the application of single particle ICP-MS and isotope dilution analysis including the development of high accuracy sample preparation procedures utilizing chemical separations. She has authored or co-authored more than 50 peer-reviewed publications describing these activities. has been working at the National Institute of Standards and Technology-Chemical Science Division-Chemical Process and Nuclear Measurements Group since 1993 in certification of more than 40 elements in various Standard Reference Materials by Instrumental Neutron Activation Analysis (INAA). received his M.S. in 1981 at Western Illinois University, and his Ph.D. in 1988 at Purdue University. He has worked as a research chemist for the National Institute of Standards and Technology since 1991, and specializes in prompt gamma-ray activation analysis and both instrumental and radiochemical neutron activation analysis. is currently responsible for NIST policies related to providing public access to results of federally funded research. For 24 years she was an analytical chemist in the Chemical Sciences Division, Material Measurement Laboratory, at NIST. is one of the program coordinators for both the food and the dietary supplements SRM programs at National Institute of Standards and Technology (NIST). She is also one of the program coordinators for the Health Management Assessment Quality Assurance Program (HAMQAP). While at NIST, she has been involved in the development and certification of numerous SRMs, primarily for nutritional and toxic elements. For the past ten years, focus has been on the development of the QAP and the food and dietary supplement SRM program. serves as a Mathematical Statistician in the Statistical Engineering Division of the Information Technology Laboratory at the National Institute of Standards and Technology. He works in statistical consulting, analysis, and computing in a variety of areas, including forensic science, information technology, and in the certification of numerous food and supplement Standard Reference Materials. is a scientist emeritus with the Chemical Sciences Division of NIST. He has been working throughout his career on development and use of neutron activation analysis (NAA) at the highest metrological level applied to the value assignment in SRMs. Research interests include the characterization of elemental species and specialized NAA procedures such as coincidence spectrometry and cold neutron prompt gamma analysis. A Standard Reference Material (SRM) of seaweed, SRM 3232 Kelp Powder (Thallus laminariae) has been developed to support food and dietary supplement measurements in compliance with the Food Safety Modernization Act (FSMA) and the Dietary Supplement Health and Education Act of 1994 (DSHEA). The material was characterized for nutritional minerals, arsenic species, isomers of vitamin K1, proximates, and toxic elements. Kelp is a rich source of vitamins and minerals, and it is an excellent source of dietary iodine. Kelp also contains a large amount of arsenic, which is toxic as inorganic species but much less so as organic species. To capture the dietary profile of kelp, certified values were issued for As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Mo, Na, Pb, and Zn. Reference values for proximates were assigned. For the first time, a certified value for iodine, reference values for isomers of vitamin K1, and reference values for arsenic species including arsenosugars were assigned in a seaweed. SRM 3232 fills a gap in Certified Reference Materials (CRMs) needed for quality assurance and method validation in the compositional measurements of kelp and similar seaweeds used as food and as dietary supplements. Graphical Absract Arsenic species and isomers of vitamin K1 were determined in the development of SRM 3232 Kelp Powder (Thallus laminariae).
Keywords: Kelp; Laminaria ; Iodine; Arsenosugar; Vitamin K1 ; SRM

is a graduate student in the Department of Chemistry at Capital Normal University. Her research is focused on the fabrication of electrochemical immunosensing interfaces. is a graduate student in the Department of Chemistry at Capital Normal University. Her research is focused on electrochemical immunoassays for tumor markers. is currently Full Professor of Physical Chemistry in the Department of Chemistry at Capital Normal University. His research interests include nanobiosensors, nanofabrication, and electrochemical biosensors. Enzyme-free catalytic amplification is of great significance for sensitive label-free electrochemical immunosensors. In this study, an enzyme-free catalytic amplification based label-free amperometric immunosensor was developed for sensitive detection of neuron-specific enolase (NSE) by use of a AuPd nanoparticle–multiwalled carbon nanotube (AuPd-MWCNT) composite, ferrocenecarboxaldehyde (Fc-CHO), and chitosan hybrid hydrogel. The intrinsic virtues of chitosan not only resulted in bioactivity of the attached antibodies and made the other component of the immunosensor easier to fix on the electrode, but also imparted abundant binding sites to the hydrogel to condense Fc-CHO to achieve the initial signal amplification. Fc-CHO, which served as an electroactive species to generate a redox response, also exhibits excellent electrocatalytic activity toward H2O2. AuPd-MWCNT composite, with enhanced peroxidase-like catalytic activity, could catalyze H2O2 to accelerate electron transfer. When H2O2 was present in the detection solution, synergetic catalysis of Fc-CHO and AuPd-MWCNT composite toward H2O2 was achieved, thus realizing enzyme-free signal amplification. On the basis of this enzyme-free signal amplification, the electrochemical immunosensing platform provided a wide linear range from 1 pg mL-1 to 100 ng mL-1, a low detection limit of 0.483 pg mL-1, and high sensitivity of 7.22 μA (log10 C NSE)-1. Moreover, the immunosensor showed enormous potential in clinical application. Graphical abstract An enzyme-free catalytic amplification based label-free amperometric immunosensor was developed for sensitive detection of neuron-specific enolase (NSE) by use of a AuPd nanoparticle–multiwalled carbon nanotube (MWCNT) composite, ferrocenecarboxaldehyde (Fc-CHO), and chitosan (CS) hybrid hydrogel. BSA bovine serum albumin, GA glutaraldehyde, SWV square wave voltammetry
Keywords: Label-free electrochemical immunosensor; Enzyme-free catalytic amplification; Ferrocenecarboxaldehyde; AuPd nanoparticle–multiwalled carbon nanotube composite; Chitosan; Neuron-specific enolase

Two complementary reversed-phase separations for comprehensive coverage of the semipolar and nonpolar metabolome by Fuad J. Naser; Nathaniel G. Mahieu; Lingjue Wang; Jonathan L. Spalding; Stephen L. Johnson; Gary J. Patti (1287-1297).
is a graduate student at Washington University in St. Louis. He is interested in improving untargeted metabolomic analyses, in terms of both biochemical coverage and throughput. is a postdoctoral researcher at Washington University in St. Louis. He is interested in developing new computational approaches to interrogate large data sets. is a graduate student at Washington University in St. Louis. His research focuses on understanding how reduction–oxidation reactions regulate cellular metabolism. is a graduate student at Washington University in St. Louis. He is developing and applying metabolomics to investigate questions in developmental biology. is a Professor of Genetics at Washington University in St. Louis. His laboratory studies growth control using a variety of techniques, including transposon-based clonal analysis, vital (green fluorescent protein expressing) markers, and metabolomics. is the Michael and Tana Powell Associate Professor in the Department of Chemistry at Washington University in St. Louis. The Patti laboratory develops NMR-based and mass-spectrometry-based metabolomic technologies that can be applied to problems in mammalian physiology. Although it is common in untargeted metabolomics to apply reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) methods that have been systematically optimized for lipids and central carbon metabolites, here we show that these established protocols provide poor coverage of semipolar metabolites because of inadequate retention. Our objective was to develop an RPLC approach that improved detection of these metabolites without sacrificing lipid coverage. We initially evaluated columns recently released by Waters under the CORTECS line by analyzing 47 small-molecule standards that evenly span the nonpolar and semipolar ranges. An RPLC method commonly used in untargeted metabolomics was considered a benchmarking reference. We found that highly nonpolar and semipolar metabolites cannot be reliably profiled with any single method because of retention and solubility limitations of the injection solvent. Instead, we optimized a multiplexed approach using the CORTECS T3 column to analyze semipolar compounds and the CORTECS C8 column to analyze lipids. Strikingly, we determined that combining these methods allowed detection of 41 of the total 47 standards, whereas our reference RPLC method detected only 10 of the 47 standards. We then applied credentialing to compare method performance at the comprehensive scale. The tandem method showed more than a fivefold increase in credentialing coverage relative to our RPLC benchmark. Our results demonstrate that comprehensive coverage of metabolites amenable to reversed-phase separation necessitates two reconstitution solvents and chromatographic methods. Thus, we suggest complementing HILIC methods with a dual T3 and C8 RPLC approach to increase coverage of semipolar metabolites and lipids for untargeted metabolomics. Graphical abstract Analysis of semipolar and nonpolar metabolites necessitates two reversed-phase chromatography (RPLC) methods, which extend metabolome coverage more than fivefold for untargeted profiling. HILIC hydrophilic interaction liquid chromatography
Keywords: Metabolomics; Untargeted profiling; Mass spectrometry; Global coverage; Semipolar metabolome; Reversed-phase

is currently a PhD candidate at the German Cancer Research Center (DKFZ) in Heidelberg, Germany. He received his bachelor’s degree in biology from the Aristotle University of Thessaloniki in Greece, followed by a one year training in bioinformatics at the European Molecular Biology Laboratory (EMBL) in Heidelberg as an Otto Bayer fellow. He is interested in host–microbe interactions and in studying RNA editing as driver of cellular diversity. is a chemistry and biology graduate with a PhD in biology, and is currently a lecturer at the Department of Biology, School of Sciences, in the Aristotle University of Thessaloniki, Greece. She has been working on environmentally friendly treatments of bacterial animal diseases and pollutants as well as the development of analytical methods for the determination of antibiotics in animal tissues. She is interested in the study of host–pathogen interactions, the discovery of novel natural antibiotics such as bacteriocins, and the study of potential applications of bacteriocins against pollutants and animal and human diseases. A novel, generally applicable method of identifying peptides using HPLC, microwave-assisted acid hydrolysis (MAAH), and bioinformatics is described. Method validation was performed on bacteriocins—antibacterial peptides produced by probiotic bacteria—using nine different bacteriocin isolates secreted by the probiotic Lactococcus lactis. Calibration curves were constructed for 23 amino acid PTH derivatives, and analysis was performed using norleucine as the internal standard. Validation of amino acid analysis performed in the range 2.5–100 nmol/mL indicated excellent method linearity, while the LODs ranged from 0.17 to 2.88 nmol/mL and the LOQs from 0.51 to 8.75 nmol/mL. The MAAH method was developed by irradiating nisaplin for various durations at 700 W, with 7 min providing the best results. The amino acid content of each sample was estimated following the application of MAAH to ten different samples. The bacteriocins in our samples were identified using the UniProt database. Eight of nine peptides were identified as UniProt entries: nisin A (P13068), nisin Z (P29559), I4DSZ9, OB7236, P36499, OB7237, A0A0M7BH60, and T2C9F0. The phylogenetic tree was constructed for nisin A and nisin Z using the multiple sequence aligning tool Clustal Ω. The identified nisin types presented excellent correlation with their ModBase-predicted structures. The present method gives true, precise, and rapid results, and requires only standard technical equipment. Our results suggest that the present approach can facilitate the discovery of novel bacteriocins and provide useful information on not only the amino acid contents of peptides but also the evolution of protein biology. Graphical abstract Identification of eight bacteriocins secreted by the probiotic L. lactis, following microwave assisted acid hydrolysis (MAAH), amino acid content analysis of each sample with HPLC-DAD and bioinformatics analysis using Uniprot, Clustal Ω and ModBase.
Keywords: Amino acids; HPLC; Microwave-assisted acid hydrolysis; Bacteriocins; Bionformatics

Quaternary ammonium salts (QAS), both linear and bicyclic, are often utilized to improve the mass spectrometry (MS) analysis of peptides by fixing a permanent positive charge on the analyzed molecule. However, during collision-induced dissociation (CID) experiments, QAS undergo unwanted side reactions—Hofmann elimination as well as a tertiary amine loss— rendering the data interpretation complicated. In this work, we present 2-thia- and 2-oxa-5-azoniaspiro[4.4]nonyl groups as heterocyclic derivatives of the highly stable ionization group, 5-azoniaspiro[4.4]nonyl, for a sensitive peptide analysis by MS. Due to the permanent positive charge, labeled peptides are characterized by enhanced ionization efficiency during electrospray mass spectrometry (ESI-MS) conditions. Moreover, interpretation of the CID fragmentation of labeled peptides is facilitated since a series of generated fragmentation ions enable a complete sequence coverage. Introduction of a heteroatom into the 5-azoniaspiro[4.4]nonyl scaffold allows for liberation of a stable reporter ion which could be used in selected reaction monitoring (SRM)-targeted quantification experiments. Additionally, we synthesized a deuterated analog of the tag for LC-SRM-targeted quantitative analysis. The obtained results indicate the general usefulness of the proposed heterocyclic quaternary ammonium ionization tag for sequencing and quantification of peptides. Graphical abstract New reagents based on the structure of the 5-azoniaspiro[4.4]nonyl tag for peptide analysis by tandem mass spectrometry
Keywords: Derivatization of peptides; Peptide sequencing; Ionization tag; Tandem mass spectrometry

Determination of total phthalate in cosmetics using a simple three-phase sample preparation method by Laping Liu; Zhengmeng Wang; Sihan Zhao; Jiahui Duan; Hu Tao; Wenji Wang; Shuhui Liu (1323-1331).
A simple sample preparation method requiring minimal organic solvents is proposed for the determination of the total phthalate content in cosmetics by high-performance liquid chromatography-tandem mass spectrometry. The hydrolysis of phthalates and purification of interfering substances were performed in a three-phase system that included an upper n-hexane phase, a middle ethanol phase, and a lower aqueous alkali solution. This three-phase system utilized an incremental purification strategy. The apolar ingredients were extracted with n-hexane, the polar pigments accumulated in the ethanol phase, and the hydrolysis product, phthalic acid, remained in the hydrolysate. Under the optimized conditions, the correlation coefficients (r) for the calibration curves were 0.998–0.999 in the range 0.60–12 mol L−1. The limit of detection was 5.1 μmol kg−1, and the limit of quantification was 9.2 μmol kg−1. The recoveries varied from 84 to 97% with RSDs equal to or lower than 11%. The intra-day and inter-day repeatability values, expressed as the relative standard deviation, were less than 8.7 and 9.8, respectively. No obvious matrix effect existed in the different cosmetics matrices. The validated method was applied for the analysis of 57 commercial cosmetic samples. Graphical abstract Analysis of phthalates in cosmetics using a three-phase preparation method
Keywords: Cosmetics; Purification; Total phthalate; Phthalic acid

Utilizing mass spectrometry imaging to map the thyroid hormones triiodothyronine and thyroxine in Xenopus tropicalis tadpoles by Naoko Goto-Inoue; Tomohiko Sato; Mizuki Morisasa; Akihiko Kashiwagi; Keiko Kashiwagi; Yuki Sugiura; Eiji Sugiyama; Makoto Suematsu; Tsukasa Mori (1333-1340).
Thyroid hormones are not only responsible for thermogenesis and energy metabolism in animals, but also have an important role in cell differentiation and development. Amphibian metamorphosis provides an excellent model for studying the remodeling of the body. This metamorphic organ remodeling is induced by thyroid hormones, and a larval body is thus converted into an adult one. The matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) imaging technology is expected to be a suitable tool for investigating small bioreactive molecules. The present study describes the distribution of the thyroid hormones, i.e., triiodothyronine (T3) and thyroxine (T4) and their inactive form reverse T3 (rT3) in Xenopus tropicalis tadpoles using two different types of imaging techniques, MS/MS and Fourier transform (FT)-MS imaging. As a result of MS/MS imaging, we demonstrated that T3 was mainly distributed in the gills. T4 was faintly localized in the eyes, inner gills, and intestine during metamorphosis. The intensity of T3 in the gills and the intensity of T4 in the body fluids were increased during metamorphosis. Moreover, the localization of the inactive form rT3 was demonstrated to be separate from T3, namely in the intestine and muscles. In addition, FT-MS imaging could utilize simultaneous imaging including thyroid hormone. This is the first report to demonstrate the molecular distribution of thyroid hormones themselves and to discriminate T3, T4, and rT3 in animal tissues.
Keywords: Thyroid hormone; Metamorphosis; Mass spectrometry imaging; Amphibian

Characterization of an easy-to-use method for the routine analysis of the central metabolism using an affordable low-resolution GC–MS system: application to Arthrospira platensis by Myriam Phélippé; Rémy Coat; Camille Le Bras; Lorene Perrochaud; Eric Peyretaillade; Delphine Kucma; Abdellah Arhaliass; Gérald Thouand; Guillaume Cogne; Olivier Gonçalves (1341-1361).
We developed an easy-to-use method for the routine analysis of the central metabolism using an affordable low-resolution GC–MS system run in SIM mode. The profiling approach was optimized for the derivatization protocol of some 60 targeted metabolites. The performance of two silylation reagents (MSTFA and BSTFA) that allowed the comprehensive derivatization of 42 key intermediary metabolites of the 60 initially targeted (organic acids, phosphate derivatives, monosaccharides and amino acids) was measured. The experimental results unequivocally showed that the MSTFA reagent met mandatory criteria including ease of handling (a very simple one-step protocol was developed), comprehensiveness of derivatization (the 42 compounds covered the extended metabolic pathways of the central carbon metabolism, with a coverage percentage ranging from 17% for the worst to 90% for the best result), optimized response coefficient of the whole derivatives (median value greater than the others by one order of magnitude) and repeatability of the protocol (RSD value below 25% for the whole procedure). When tested in real conditions (cyanobacteria polar extract), the experimental results showed that the profiling methodology was adequately repeatable (RSD = 35%) to ensure quantification results comparable with much more sensitive analytical techniques (capillary electrophoresis/mass spectrometry and liquid chromatography/triple quadrupole mass spectrometry system), while needing only about twice the quantity of biomass. Graphical abstract Schematic overview of an easy-to-use profiling method for the routine analysis of the central metabolism using a low-resolution GC-MS system
Keywords: Gas chromatography–mass spectrometry; Central carbon metabolism; Elementary bricks; Metabolic precursors; Intermediary metabolites; Cyanobacteria

Brassinosteroids (BRs) are important plant hormones regulating plant growth and development. High-performance analytical methods for quantifying endogenous BRs are important for studying the molecular mechanisms of BR action. Herein we developed a high-performance sample pretreatment method based on boronate affinity magnetic nanoparticles (BAMNPs). The high specificity of boronate affinity enables direct fishing of BRs from plant matrices. The strong binding energy makes it possible to remove most contaminants in plant matrices with a small loss of target BRs. Besides these advantages, the novel two-step oxidation-hydrolysis elution system raised BR recoveries to 70.5%–98.2%, which was much higher than other boronate affinity applications. The high cleanliness of the final eluents lowered the matrix effects to 85.2%–92.4%. As a result, this method enables simultaneously good recoveries of endogenous BRs and thorough removal of matrix interferences, which greatly improves the sensitivity of BR analysis and reduces the use of plant materials for routine analysis to <10 mg. In addition, the sample handling time can be shortened to <3 h due to the operating convenience of BAMNPs and their easy separation from plant powders. Based on these advantages of BAMNP solid phase extraction, the organ-specific BR distribution analysis in Arabidopsis and rice tissues demonstrates excellent sensitivity, good reproducibility and high throughput of the method. Graphical abstract A high-sensitivity and time-saving UPLC-MS/MS-based quantification method for brassinosteroids (BRs) was developed through directly fishing BRs from plant matrices and eliminating most matrix interferences with as-prepared boronate affinity magnetic nanoparticles (BAMNPs).
Keywords: Boronate affinity magnetic nanoparticles; Solid phase extraction; Ultratrace analysis; Brassinosteroids; Liquid chromatography tandem mass spectrometry; Matrix effect

Determination of halogenated flame retardants by GC-API-MS/MS and GC-EI-MS: a multi-compound multi-matrix method by Frank Neugebauer; Annekatrin Dreyer; Nina Lohmann; Jan Koschorreck (1375-1387).
The extensive application of halogenated flame retardants has led to their widespread distribution in the environment. Recently, concerns emerged regarding their potential persistence, (bio)accumulation, and/or toxicity. Particularly halogenated flame retardants based on norbornene structures, like Dechlorane Plus as well as other brominated PBDE replacements, generically called emerging, novel, or alternative flame retardants, are in the focus of interest. A comprehensive analytical method for the determination of 21 halogenated flame retardants (HFRs) of different substance classes (dechloranes, brominated aromates, brominated ethers, cyclic BFR) in a broad variety of matrices (tree leaves, fish fillet, birds eggs, suspended particles) was developed in order to assess their environmental levels as well as temporal trends, especially for the use within environmental specimen banks (ESBs). In addition to the alternative HFRs, a set of 24 PBDEs were measured in the same samples, however using GC-EI-MS for detection. Samples were extracted using accelerated solvent extraction (ASE) with dichloromethane:hexane (exception: soxhlet extraction for suspended particles) followed by a multi column clean-up. Quantification was performed by API-GC-MS/MS as a modern, gentle, and sensitive technique for simultaneous detection of compounds throughout a wide range of masses and fragmentation characteristics (exception: PBDE detection using GC-EI-MS). With the exception of BDE 209, instrumental precisions of target compounds ranged from 1% to 16 % (at levels of 2 pg injection–1 for HFR, 20 pg injection–1 for DBDPE, 7-36 pg injection–1 for PBDEs). Interday precisions of the entire analytical method including extraction and clean-up were mostly below 25% for all validation matrices at spiked levels of 100 pg sample–1 for HFR (DBDPE: 1000 pg sample–1) and 1200–6000 pg sample–1 for PBDEs. The majority of analytes were investigated with expanded measurement uncertainties of less than 50%. Graphical abstract A new approach to HFR analysis in multiple environmental matrices
Keywords: Dechlorane Plus; Brominated flame retardants; nBFR; German environment specimen bank; Atmospheric pressure ionization

Lactoferrin (LF) is an important multifunctional protein that comprises a large fraction of the protein mass in certain human fluids and tissues, and its concentration is often used to assess health and disease. LF can be nitrated by multiple routes, leading to changes in protein structure, and nitrated proteins can negatively impact physiological health via nitrosative stress. Despite an awareness of the detrimental effects of nitrated proteins and the importance of LF within the body, cost-effective methods for detecting and quantifying nitrated lactoferrin (NLF) are lacking. We developed a procedure to selectively quantify NLF using sandwich enzyme-linked immunosorbent assay (ELISA), utilizing a polyclonal anti-LF capture antibody paired with a monoclonal anti-nitrotyrosine detector antibody. The assay was applied to quantify NLF in samples of pure LF nitrated via two separate reactions at molar ratios of excess nitrating agent to the total number of tyrosine residues between 10/1 and 100/1. Tetranitromethane (TNM) was used as a laboratory surrogate for an environmental pathway selective for production of 3-nitrotyrosine, and sodium peroxynitrite (ONOO) was used as a surrogate for an endogenous nitration pathway. UV-vis spectroscopy (increased absorbance at 350 nm) and fluorescence spectroscopy (emission decreased by > 96%) for each reaction indicate the production of NLF. A lower limit of NLF detection using the ELISA method introduced here was calculated to be 0.065 μg mL−1, which will enable the detection of human-physiologically relevant concentrations of NLF. Our approach provides a relatively inexpensive and practical way to assess NLF in a variety of systems. Graphical abstract We developed a procedure to selectively quantify nitrated lactoferrin (NLF) protein using a sandwich enzyme-linked immunosorbent assay (ELISA) and verified results against several spectroscopic techniques. Our approach provides an inexpensive and practical way to assess NLF in a variety of systems.
Keywords: Protein nitration; Nitrotyrosine; Ocular disease; Atmospheric pollution; Peroxynitrite; Tetranitromethane

We developed a highly sensitive and selective sensor based on the nanoprobe conjugates of catalytic nanoparticles and double-stranded DNA (dsDNA) for the colorimetric detection of NF-κB protein. The sensing mechanism takes advantage of the catalytic activity of nanoparticle surfaces and the specific binding of NF-κB to a dsDNA sequence. In the presence of NF-κB, the highly selective interactions between dsDNA and NF-κB lead to the passivation of the catalytic nanoparticle surfaces, impeding the sodium borohydride-mediated reduction rate of 4-nitrophenol. The correlation between the NF-κB concentration and the visualized reduction rate of 4-nitrophenol from yellow to colorless clearly demonstrates the highly quantitative nature of the sensor. Importantly, this sensor can conclusively detect concentrations as low as 6.39 pM of NF-κB, which to best of our knowledge is the lowest limit of detection for a colorimetric NF-κB detection system. The excellent sensitivity of this sensor relies on the high binding constant of NF-κB to dsDNA and the catalytic activity of nanoparticle surfaces for the signal amplification. This sensor allows visual detection without the need for any spectrometric instrumentation. We also determined the various parameters such as the pH, temperature, incubation time, and salt concentration for optimal NF-κB-dsDNA interactions. Finally, we demonstrated the performance of the sensor with simulated sample analysis. Graphical abstract A highly sensitive and selective colorimetric detection of protein NF-κB using the nanoprobeconjugates of catalytic gold nanoparticles and double-stranded DNA (dsDNA) has been developed.
Keywords: Gold nanoparticle; DNA; Colorimetric detection; Protein