Analytical and Bioanalytical Chemistry (v.408, #11)

Meet the Guest Editors by Nicola Oberbeckmann-Winter (2631-2633).

Meet the Contributors by Nicola Oberbeckmann-Winter (2635-2640).

Mercury (Hg) pollution is considered a major environmental problem due to the extreme toxicity of Hg. However, Hg metabolic pathways in biota remain elusive. An understanding of these pathways is crucial to elucidating the (eco)toxic effects of Hg and its biogeochemical cycle. The development of a new analytical methodology based on both speciation and natural isotopic fractionation represents a promising approach for metabolic studies of Hg and other metal(loid)s. Speciation provides valuable information about the reactivity and potential toxicity of metabolites, while the use of natural isotopic signature analysis adds a complementary dynamic dimension that allows the life history of the target element to be probed, the source of the target element (i.e., the source of pollution) to be identified, and reactions to be tracked. The resulting combined (bio)molecular and isotopic signature affords precious insight into the behavior of Hg in biota and Hg detoxification mechanisms. In the long term, this highly innovative methodology could be used in life and environmental science studies of metal(loid)s to push back the frontiers of our knowledge in this field. This paper summarizes the current status of the application of Hg speciation and the isotopic signature of Hg at the biomolecular level in living organisms, and discusses potential future uses of this combination of techniques. Graphical Abstract Application of Hg speciation and the isotopic signature of Hg to enhance our understanding of the roles of Hg in metabolic, toxicological, and environmental processes
Keywords: Biological samples; Metals; Heavy metals; Speciation; Isotopic fractionation; Mercury

Because noncovalent interface functionalization is frequently required in graphene-based devices, biomolecular self-assembly has begun to emerge as a route for controlling substrate electronic structure or binding specificity for soluble analytes. The remarkable diversity of structures that arise in biological self-assembly hints at the possibility of equally diverse and well-controlled surface chemistry at graphene interfaces. However, predicting and analyzing adsorbed monolayer structures at such interfaces raises substantial experimental and theoretical challenges. In contrast with the relatively well-developed monolayer chemistry and characterization methods applied at coinage metal surfaces, monolayers on graphene are both less robust and more structurally complex, levying more stringent requirements on characterization techniques. Theory presents opportunities to understand early binding events that lay the groundwork for full monolayer structure. However, predicting interactions between complex biomolecules, solvent, and substrate is necessitating a suite of new force fields and algorithms to assess likely binding configurations, solvent effects, and modulations to substrate electronic properties. This article briefly discusses emerging analytical and theoretical methods used to develop a rigorous chemical understanding of the self-assembly of peptide–graphene interfaces and prospects for future advances in the field.
Keywords: Graphene; Peptide; Scanning tunneling microscopy; Atomic force microscopy; Polarization modulation IR reflection–absorption spectroscopy; Quartz crystal microgravimetry

Bioanalytical approaches for the detection of protein acetylation-related enzymes by Pei Li; Yitao Han; Yong Li; Rong Zhu; Huixia Wang; Zhou Nie; Shouzhuo Yao (2659-2668).
Reversible protein acetylation catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) is an essential post-translational modification (PTM) mechanism which correlates largely with epigenetic gene regulation such as transcriptional activation, DNA replication, histone deposition, and DNA repair. Dysfunction of histone acetylation and the aberrant activity of HATs/HDACs is often associated with the pathogenesis of numerous diseases, especially cancer. Therefore, developing potent and specific analytical methods for HATs/HDACs is important for fundamental biochemical research, disease diagnosis and treatment, and drug development. This paper briefly summarizes the general design strategies used in HAT/HDAC sensors, gives a systematic overview of recent advances in the analytical methods for HAT/HDAC enzymatic analysis, classifies these methods by their biorecognition mechanisms and relative applications either in vitro or in living cells, then outlines challenges faced by these bioanalytical methods and offers perspectives on future developments. Graphical Abstract Reversible acetylation modification process and the general sensing mechanisms of protein acetylation-related enzymes (PAREs) activity
Keywords: Protein acetylation; Acetyltransferase and deacetylase; Analytical method; Drug screening

This paper gives a critical overview of capillary electrophoresis (CE) methodologies recently developed for controlling and optimizing the synthesis of nanoparticles as well as characterizing their functionalization in terms of physicochemical properties. Thanks to their electrophoretic mobility, various parameters can be determined, such as NP size and charge distribution, ζ-potential, surface functionality, colloidal stability, grafting rates, and dissociation constants, allowing not only the complete characterization of new nanoprobes but also helping in their design and in the selection of chemical conditions for their storage and further manipulation. New strategies for the improvement of CE detection sensitivity are also described. Graphical Abstract Electrokinetic methodologies can yield deeper insights into the world of nanoparticles
Keywords: Capillary electrophoresis; Nanoparticle design; Physicochemical characterization; Surface chemistry; Biocompatibility; Stability; Sensitivity

Recent trends in the analysis of bioactive peptides in milk and dairy products by Anna Laura Capriotti; Chiara Cavaliere; Susy Piovesana; Roberto Samperi; Aldo Laganà (2677-2685).
Food-derived constituents represent important sources of several classes of bioactive compounds. Among them peptides have gained great attention in the last two decades thanks to the scientific evidence of their beneficial effects on health in addition to their established nutritional value. Several functionalities for bioactive peptides have been described, including antioxidative, antihypertensive, anti-inflammatory, immunomodulatory, and antimicrobial activity. They are now considered as novel and potential dietary ingredients to promote human health, though in some cases they may also have detrimental effects on health. Bioactive peptides can be naturally occurring, produced in vitro by enzymatic hydrolysis, and formed in vivo during gastrointestinal digestion of proteins. Thus, the need to gain a better understanding of the positive health effects of food peptides has prompted the development of analytical strategies for their isolation, separation, and identification in complex food matrices. Dairy products and milk are potential sources of bioactive peptides: several of them possess extra-nutritional physiological functions that qualify them to be classified under the functional food label. In this trends article we briefly describe the state-of-the-art of peptidomics methods for the identification and discovery of bioactive peptides, also considering recent progress in their analysis and highlighting the difficulty in the analysis of short amino acid sequences and endogenous peptides.
Keywords: Bioactive peptides; Dairy products; Milk; Mass spectrometry; Peptidomic analysis

We describe trends in fast, high resolution elemental imaging by laser ablation–inductively coupled plasma mass spectrometry (LA–ICPMS). Recently developed low dispersion LA cells deliver quantitative transport of ablated aerosols within 10 ms and also provide enhanced sensitivity compared to conventional LA cells because the analyte ion signal becomes less diluted during aerosol transport. When connected to simultaneous ICPMS instruments, these low dispersion LA cells offer a platform for high speed and high lateral resolution shot-resolved LA–ICPMS imaging. Here, we examine the current paradigms of LA–ICPMS imaging and discuss how newly developed LA cell technology combined with simultaneous ICPMS instrumentation is poised to overcome current instrumental limitations to deliver faster, higher resolution elemental imaging. Graphical Abstract On means for obtaining high-speed, high-resolution, multielemental images is to combine new lowdispersion laser-ablation cell technology with an inductively coupled plasma time-of-flight mass spectrometer (ICP-TOFMS). Here, we show three selected-isotope LA-ICP-TOFMS images of a hetereogeneous Opalinus clay sample
Keywords: Laser ablation; ICPMS; Elemental imaging; Mass spectrometry; Time-of-flight

Nanoparticle chains as electrochemical sensors and electrodes by Long Pu; Maarij Baig; Vivek Maheshwari (2697-2705).
With the advances in the field of nanotechnology, significant progress is being achieved in fabrication of nanoscale electrodes (nanoelectrodes) and using their properties for applications in multiple fields. Compared with conventional macroscale electrodes, nanoelectrodes offer many advantages that arise from their limited size. Self-assembled chains of metal nanoparticles in particular have drawn interest for fabrication of nanoelectrodes because of their unique electrical properties and geometric morphology. This article discusses the fabrication methods and potential applications of nanoparticle chains as nanoelectrodes in electrochemical systems and also as conductometric sensors. The challenges for such systems are also summarized.
Keywords: Biosensors; Electroanalytical methods; Electrochemical sensors; Nanoparticles/nanotechnology

Many modern energy storage technologies operate via the nominally reversible shuttling of alkali ions between an anode and a cathode capable of hosting them. The degradation process that occurs with normal usage is not yet fully understood, but emerging progress in analytical tools may help address this knowledge gap. By interrogating ionic fluxes over electrified surfaces, scanning probe methods may identify features that impact the local cyclability of a material and subsequently help inform rational electrode design for future generations of batteries. Methods developed for identifying ion fluxes for batteries show great promise for broader applications, including biological interfaces, corrosion, and catalysis. Graphical Abstract Versatile ionics for next-generation batteries
Keywords: Electroanalytical methods; Electrochemical sensors; Li-ion batteries; SECCM; SECM; SICM; Stripping analysis

Ion-selective optical sensing is an important branch of analytical and bioanalytical chemistry. Conventional ion-selective optodes are based on H+ chromoionophores. These sensors are known to be pH dependent and usually operated in a passive mode. In view of the applications in complex real samples, the sensors must exhibit not only excellent chemical selectivity but also the ability to eliminate the optical background interference such as autofluorescence and light scattering. In this article, recent advances to renovate the chromoionophores and detection modes to overcome the pH cross-response and to eliminate the background optical interference are summarized. Topics include sensors based on solvatochromic dyes, alternative chromoionophores, photoswitchable sensors, upconverting nanoparticles, luminescence decay time, and others. Graphical Abstract Ion-selective optical sensing is an important branch of analytical and bioanalytical chemistry. In view of the applications in complex real samples, this article highlighted recent advances to renovate the chromoionophores and detection modes to overcome the drawbacks from the pH cross-response and background optical interference
Keywords: Ionophore; Solvatochromic dyes; Spiropyran; Photoswitching; Upconverting; Luminescence decay time

Nanosensors for neurotransmitters by Elena Polo; Sebastian Kruss (2727-2741).
Neurotransmitters are an important class of messenger molecules. They govern chemical communication between cells for example in the brain. The spatiotemporal propagation of these chemical signals is a crucial part of communication between cells. Thus, the spatial aspect of neurotransmitter release is equally important as the mere time-resolved measurement of these substances. In conclusion, without tools that provide the necessary spatiotemporal resolution, chemical signaling via neurotransmitters cannot be studied in greater detail. In this review article we provide a critical overview about sensors/probes that are able to monitor neurotransmitters. Our focus are sensing concepts that provide or could in the future provide the spatiotemporal resolution that is necessary to ‘image’ dynamic changes of neurotransmitter concentrations around cells. These requirements set the bar for the type of sensors we discuss. The sensor must be small enough (if possible on the nanoscale) to provide the envisioned spatial resolution and it should allow parallel (spatial) detection. In this article we discuss both optical and electrochemical concepts that meet these criteria. We cover techniques that are based on fluorescent building blocks such as nanomaterials, proteins and organic dyes. Additionally, we review electrochemical array techniques and assess limitations and possible future directions.
Keywords: Nanosensors; Fluorescent probes; Neurotransmitters; Chemical imaging; Electrochemistry

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

The detection of cancer at an early stage is often significant in the successful treatment of the disease. Tumor cells have been reported to generate unique cancer volatile organic compound (VOC) profiles which can reflect the disease conditions. The detection and analysis of VOC biomarkers from exhaled breath has been recognized as a new frontier in cancer diagnostics and health inspections owing to its potential in developing rapid, noninvasive, and inexpensive cancer screening tools. To detect specific VOCs of low concentrations from exhaled breath, and to enhance the accuracy of early diagnosis, many breath collection and analysis approaches have been developed. This paper will summarize and critically review the exhaled-breath VOC-related sampling, collection, detection, and analytical methods, especially the recent development in VOC sensors. VOC sensors are commonly inexpensive, portable, programmable, easy to use, and can obtain data in real time with high sensitivities. Therefore, many sensor-based VOC detection techniques have huge potential in clinical point-of-care use.
Keywords: Volatile organic compounds; Cancer diagnosis; Exhaled breath; Sensors

Fingerprints have long been and are still considered to be the gold standard for personal identification in forensic investigations. Developing or enhancing the visualization of invisible fingerprints, so-called latent fingerprints (LFPs), remains to be the core subject in forensic science. Moreover, the past few years have witnessed a renewal of research interest in the possibility that a fingerprint can provide additional information than just identification of individuals, such as personal traits, the presence of human metabolites with diagnostic values, and the evidence of contact with explosives or illicit drugs. Fingerprint analysis has manifested itself as a research area far beyond the scope of forensics, to which not only conventional fingerprint examiners but also researchers from chemistry, biochemistry, medical science, material science, and nanotechnology fields have made significant contributions in recent years. Beginning with a brief overview of the components present in LFP residue that essentially determines which method or reagent will give the best visualization result, this paper reviews the progress since 2007 on new reagents and methods developed for LFP visualization and simultaneous detection of specific chemicals present in the LFP residue, with an emphasis on the utilization of mass spectrometry, infrared spectroscopy, nanoparticles, and immunogenic and nucleic acid reagents. Graphical Abstract Fingerprint visualization and analysis by imaging ridge residue components.
Keywords: Latent fingerprints; Forensic science; Visualization; Component recognition; Analytical chemistry

Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges by Taiping Qing; Dinggeng He; Xiaoxiao He; Kemin Wang; Fengzhou Xu; Li Wen; Jingfang Shangguan; Zhengui Mao; Yanli Lei (2793-2811).
Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as ‘nano-tools’ for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors. Graphical abstract Nucleic acid tool enzymes-aided signal amplification sensing
Keywords: Nucleic acid tool enzymes; Signal amplification; Nucleic acid; Aptamer; Sensing

Non-spherical noble metal nanoparticles (NPs) have widely tunable localized surface plasmon resonance, very high extinction coefficient, and strongly facet-dependent adsorption/binding properties. A few non-spherical noble metal NPs have been employed as reporters and/or modulators for various optical sensing. This review summarizes recent progress in the study of design, performance, and application of colorimetric and fluorescent sensing/biosensing systems based on three kinds of non-spherical noble metal NPs with different dimension, namely, one- (or quasi-one) dimensional nanorods, two-dimensional nanoplates, and three-dimensional nanodendritics; furthermore, the future developments in this research area are also discussed.
Keywords: Sensing; Biosensing; Optical sensors; Non-spherical metal nanoparticles

The future point-of-care detection of disease and its data capture and handling by Natalia Lopez-Barbosa; Jorge D. Gamarra; Johann F. Osma (2827-2837).
Point-of-care detection is a widely studied area that attracts effort and interest from a large number of fields and companies. However, there is also increased interest from the general public in this type of device, which has driven enormous changes in the design and conception of these developments and the way data is handled. Therefore, future point-of-care detection has to include communication with front-end technology, such as smartphones and networks, automation of manufacture, and the incorporation of concepts like the Internet of Things (IoT) and cloud computing. Three key examples, based on different sensing technology, are analyzed in detail on the basis of these items to highlight a route for the future design and development of point-of-care detection devices and their data capture and handling. Graphical Abstract The future Point-of-care device key elements: recognition element, end user device and integration to other technological platforms and manufacture processes
Keywords: Biochips; High - throughput screening; Biosensors; Biotechnological products; Clinical; Biomedical analysis; Electrochemical sensors; Mass sensitive sensors; Process analysis

Recent development and application of cataluminescence-based sensors by Zi Long; Hong Ren; Yuhan Yang; Jin Ouyang; Na Na (2839-2859).
A cataluminescence (CTL)-based sensor is fabricated based on the CTL signals generated from catalytic reaction on the surface of solid catalytic materials. CTL-based sensors have been developed since the 1990s and have attracted extensive attention due to long-term stability, linear concentration dependence, good reproducibility and fast response. In recent years, CTL-based sensors and sensor arrays have played important roles in chemical analysis, and were applied to determine the presence of organic gas, inorganic gas, or biological molecules, or to evaluate catalysts. However, due to the relatively low catalytic ability of catalysts or low reactivity of some analytes, high working temperature was normally adopted, which limited the applications. Recently, more advanced techniques were introduced into the fabrication of CTL-based sensors to increase the range of applications, such as advanced enrichment techniques, advanced sampling methods, advanced assisted devices, or multiple detections in array or tandem forms. This review summarizes the recent advancements of CTL-based sensors on development of advanced equipment, advanced sensing materials, new working principles examination, and new applications. Finally, we discuss some critical challenges and prospects in this field. Graphical Abstract The development of cataluminescence-based sensor
Keywords: Cataluminescence (CTL); Sensor; Enrichment; Room-temperature sensing; Plasma-assisted sensor array; Tandem CTL

Carbon nanomaterials have attracted great interest over past decades owing to their unique physical properties, versatile functionalization chemistry, and biological compatibility. In this article, we review recent progress in application of carbon nanomaterials in laser desorption/ionization mass spectrometry (LDI MS). Various types of carbon nanomaterials, including fullerenes, carbon nanotubes, graphene, carbon nanodots, nanodiamond, nanofibers, nanohorns, and their derivative forms, are involved. The applications of these materials as new matrices or probes in matrix-assisted or surface-enhanced laser desorption/ionization mass spectrometry (MALDI or SELDI MS) are discussed. Finally, we summarize current challenges and give our perspectives on the future of applications of carbon nanomaterials in LDI MS. Graphical Abstract Carbon nanomaterials (e.g., fullerenes, carbon nanotubes, graphene, nanodiamond, etc.) can be used as novel matrices or probes in MALDI or SELDI MS
Keywords: Carbon nanomaterial; Fullerene; Nanotube; Graphene; Laser desorption/ionization; Mass spectrometry

Mid-infrared spectroscopy for protein analysis: potential and challenges by Ángela I. López-Lorente; Boris Mizaikoff (2875-2889).
Mid-infrared (MIR) spectroscopy investigates the interaction of MIR photons with both organic and inorganic molecules via the excitation of vibrational and rotational modes, providing inherent molecular selectivity. In general, infrared (IR) spectroscopy is particularly sensitive to protein structure and structural changes via vibrational resonances originating from the polypeptide backbone or side chains; hence information on the secondary structure of proteins can be obtained in a label-free fashion. In this review, the challenges for IR spectroscopy for protein analysis are discussed as are the potential and limitations of different IR spectroscopic techniques enabling protein analysis. In particular, the amide I spectral range has been widely used to study protein secondary structure, conformational changes, protein aggregation, protein adsorption, and the formation of amyloid fibrils. In addition to representative examples of the potential of IR spectroscopy in various fields related to protein analysis, the potential of protein analysis taking advantage of miniaturized MIR systems, including waveguide-enhanced MIR sensors, is detailed. Graphical Abstract A diagnosis of the potential and challenges of mid-infrared spectroscopy for protein analysis is given in this review
Keywords: Mid-infrared; Infrared spectroscopy; Proteins; Secondary structure; Amyloid; Conformational changes

Ph.D., is an NSERC Visiting Research Fellow in the department of Environmental Health Science and Research Bureau, Health Canada. Her current research is focused on the study of gene expression and epigenetics (DNA methylation, histone modifications, and miRNA expression) in mammals upon exposure to environmental chemicals. Ph.D., is currently employed as a mass spectrometrist at the University of Ottawa. His major research interests lie in the area of mass spectrometry assisted bio-marker discovery and characterization. Ph.D., is an Associate Professor of Chemistry at the University of Ottawa in Canada and a Director of Imaging and Proteomics core facilities. He won a 2015 University of Ottawa’s Young Researcher Award and was the recipient of an Early Research Award from the Ministry of Research and Innovation in 2012. His research focuses on the study of affinity interactions and conformational dynamics of biomolecules by kinetic capillary electrophoresis and mass spectrometry and on the development of DNA aptamers for sensing pathogens and cancer cells. MicroRNA molecules (miRNAs) are a class of small, single-stranded, non-coding RNA molecules that regulate cellular messenger RNA and their corresponding proteins. Extracellular miRNAs circulate in the bloodstream inside exosomes or in complexes with proteins and lipoproteins. The miRNA sequences and their quantitative levels are used as unique signatures associated with cancer diagnosis and prognosis after anticancer treatment. MicroRNAs are modified through a series of processing events after transcription like 5’-end phosphorylation, 3’- end adenylation or uridylation, terminal nucleotide deletion. The problem is that existing bioanalytical methods such as microarrays and a quantitative polymerase chain reaction are sensitive, but not capable of identifying the post-transcriptional modifications of miRNA. Here we report a capillary electrophoresis-mass spectrometry (CE-MS) method, which performs a multiplex, direct analysis of miRNAs from biological samples. Using the CE-MS method, we detected two endogenous human circulating miRNAs, a 23-nucleotide long 5’-phosporylated miRNA with 3’-uridylation (iso-miR-16-5p), and a 22-nucleotide long 5’-phosporylated miRNA (miR-21-5p) isolated from B-cell chronic lymphocytic leukemia serum. The CE separation and following MS analysis provides label-free quantitation and reveals modifications of miRNAs. MicroRNA profiling of serum samples with CE-MS has the potential to be a versatile and minimally invasive bioassay that could lead to better clinical diagnostics and disease treatment.
Keywords: Bioassays; Capillary electrophoresis/electrophoresis; MicroRNA; Serum; Direct Detection; Mass spectrometry

received her B.S. in Environmental Science from Nankai University in 2012, and next joined Dr. Huiwang Ai’s research group as a graduate student in the Environmental Toxicology Program at UCR. Her research interests focus on development of fluorescent probes for redox signaling and oxidative stress. is an Assistant Professor in the Department of Chemistry at the University of California, Riverside (UCR). Huiwang received his B.S. in Chemistry from Tsinghua University in 2003 and his Ph.D. in Chemical Biology from University of Alberta in 2008. After his post-doctoral training at The Scripps Research Institute, he joined UCR in 2011. His research program spans from development of fluorescent probes and peptide-based enzyme inhibitors to understanding of oxidative/nitrosative stress in pathophysiology. Huiwang received several awards, including the NSF CAREER Award (2014) and the Hellman Fellows Award (2013). We recently reported a redox-sensitive red fluorescent protein, rxRFP1, which is one of the first genetically encoded red-fluorescent probes for general redox states in living cells. As individual cellular compartments have different basal redox potentials, we hereby describe a group of rxRFP1 mutants, showing different midpoint redox potentials for detection of redox dynamics in various subcellular domains, such as mitochondria, the cell nucleus, and endoplasmic reticulum (ER). When these redox probes were expressed and subcellularly localized in human embryonic kidney (HEK) 293 T cells, they responded to membrane-permeable oxidants and reductants. In addition, a mitochondrially localized rxRFP1 mutant, Mito-rxRFP1.1, was used to detect mitochondrial oxidative stress induced by doxorubicin—a widely used cancer chemotherapy drug. Our work has expanded the fluorescent protein toolkit with new research tools for studying compartmentalized redox dynamics and oxidative stress under various pathophysiological conditions. Graphical abstract Novel redox-sensitive red fluorescent proteins showing a range of midpoint redox potentials
Keywords: Fluorescent protein; Oxidative stress; Genetically encoded probe; Fluorescence imaging; Compartmentalized redox dynamics

is currently pursuing a Ph.D. degree in chemistry at the University of British Columbia. She is a recipient of an NSERC Alexander Graham Bell Canada Graduate Scholarship and a UBC Four Year Doctoral Fellowship (4YF). Ms. Petryayeva received a B.Sc. (2011) degree in forensic chemistry and an M.Sc. (2012) degree in chemistry from the University of Toronto. Her major research interests include understanding the nanoparticle interface and its interactions with components of complex biological matrices, and the development of bioassays for point-of-care diagnostics using the unique optical properties of nanomaterials. is an Assistant Professor in the Department of Chemistry at the University of British Columbia. He is currently a Canada Research Chair (Tier 2) in Bio/Chemical Sensing and a Michael Smith Foundation for Health Research Scholar. Algar received B.Sc. (2005), M.Sc. (2006), and Ph.D. (2010) degrees from the University of Toronto. His group is interested in the development of luminescent materials as tools for bioanalysis, including prospective point-of-care diagnostic technologies, multifunctional cellular probes, molecular photonic logic, and biophysical and bioconjugate chemistry to support these research endeavors. Point-of-care (POC) diagnostic technologies are needed to improve global health and smartphones are a prospective platform for these technologies. While many fluorescence or photoluminescence-based smartphone assays have been reported in the literature, common shortcomings are the requirement of an excitation light source external to the smartphone and complicated integration of that excitation source with the smartphone. Here, we show that the photographic flash associated with the smartphone camera can be utilized to enable all-in-one excitation and imaging of photoluminescence (PL), thus eliminating the need for an excitation light source external to the smartphone. A simple and low-cost 3D-printed accessory was designed to create a dark environment and direct excitation light from the smartphone flash onto a sample. Multiple colors and compositions of semiconductor quantum dot (QD) were evaluated as photoluminescent materials for all-in-one smartphone excitation and imaging of PL, and these were compared with fluorescein and R-phycoerythrin (R-PE), which are widely utilized molecular and protein materials for fluorescence-based bioanalysis. The QDs were found to exhibit much better brightness and have the best potential for two-color detection. A model protein binding assay with a sub-microgram per milliliter detection limit and a Förster resonance energy transfer (FRET) assay for proteolytic activity were demonstrated, including imaging with serum as a sample matrix. In addition, FRET within tandem conjugates of a QD donor and fluorescent dye acceptor enabled smartphone detection of dye fluorescence that was otherwise unobservable without the QD to enhance its brightness. The ideal properties of photoluminescent materials for all-in-one smartphone excitation and imaging are discussed in the context of several different materials, where QDs appear to be the best overall material for this application. Graphical Abstract Bioanalytical assays with a smartphone and 3D-printed accessory for imaging photoluminescence from quantum dots
Keywords: Point-of-care diagnostics; Quantum dots; Fluorescence; FRET; Smartphone; 3D printing

Microchamber arrays with an integrated long luminescence lifetime pH sensor by Elisabeth Poehler; Simon A. Pfeiffer; Marc Herm; Michael Gaebler; Benedikt Busse; Stefan Nagl (2927-2935).
A pH probe with a microsecond luminescence lifetime was obtained via covalent coupling of 6-carboxynaphthofluorescein (CNF) moieties to ruthenium-tris-(1,10-phenanthroline)2+. The probe was covalently attached to amino-modified poly-(2-hydroxyethyl)methacrylate (pHEMA) and showed a pH-dependent FRET with luminescence lifetimes of 681 to 1260 ns and a working range from ca. pH 6.5 to 9.0 with a pKa of 7.79 ± 0.14. The pH sensor matrix was integrated via spin coating as ca. 1- to 2-μm-thick layer into “CytoCapture” cell culture dishes of 6 mm in diameter. These contained a microcavity array of square-shaped regions of 40 μm length and width and 15 μm depth that was homogeneously coated with the pH sensor matrix. The sensor layer showed fast response times in both directions. A microscopic setup was developed that enabled imaging of the pH inside the microchamber arrays over many hours. As a proof of principle, we monitored the pH of Escherichia coli cell cultures grown in the microchamber arrays. The integrated sensor matrix allowed pH monitoring spatially resolved in every microchamber, and the differences in cell growth between individual chambers could be resolved and quantified. Graphical abstract A pH probe with a microsecond luminescence lifetime is described and its covalent attachment to a hydrogel matrix, integration into microchamber arrays, and use for pH monitoring in a model E. coli miniaturized cell culture.
Keywords: Long-lifetime luminescent pH sensor; FRET-based pH imaging; Polymeric microcavity array; Escherichia coli cell growth; Bioprocess miniaturization

Pentavalent antimony uptake pathway through erythrocyte membranes: molecular and atomic fluorescence approaches by Camila Barrera; Silvana López; Luis Aguilar; Luis Mercado; Manuel Bravo; Waldo Quiroz (2937-2944).
Previous studies by our group have shown that Sb(V) is able to enter red blood cells in a dynamic process and is reduced to Sb(III) by glutathione. The present study aims to investigate a possible entry pathway for Sb(V) through the erythrocyte membrane. Applying fluorescence spectroscopy studies with Laurdan and diphenylhexatriene (DPH) probes, it was found that there was no interaction between Sb(V) and membrane lipids. By comparing the Sb(V) entry percentages through lipid vesicles and sealed erythrocyte membranes, it was found that Sb(V) required protein channels to pass through the membrane. The competitive inhibition results using HCO3 and Cl showed that the Sb(V) uptake rate through the membrane fell approximately 50–70 % until full inhibition was reached, which was possibly due to the inhibition of the anion exchanger 1 (AE1) channel. Finally, the fluorescence measurements with the 5-iodoacetamidofluorescein (5-IAF) probe showed that Sb(V) interacted with membrane protein SH groups during this process.
Keywords: Antimony; AE1; Erythrocytes; Membrane; Uptake pathway

Plastics are complex mixtures consisting of a polymer and additives with different physico-chemical properties. We developed a broad screening method to elucidate the nature of compounds present in plastics used in electrical/electronic equipment commonly found at homes (e.g., electrical adaptors, computer casings, heaters). The analysis was done by (a) solvent extraction followed by liquid chromatography coupled to high accuracy/resolution time-of-flight mass spectrometry (TOFMS) with different ionization sources or (b) direct analysis of the solid by ambient mass spectrometry high accuracy/resolution TOFMS. The different ionization methods showed different selectivity and sensitivity for the different compound classes and were complementary. A variety of antioxidants, phthalates, UV filters, and flame retardants were found in most samples. Furthermore, some recently reported impurities or degradation products derived from flame retardants were identified, such as hydroxylated triphenyl phosphate and tetrabromobisphenol A monoglycidyl ether. Graphical abstract Wide screening of plastic additives by direct probe injection (DIP)-APCI, LC-APCI and LC-ion booster ESI
Keywords: Plastics; Screening; Additives; Flame retardants; Mass spectrometry; Direct probe injection

In this work, we present a direct one-step strategy for rapidly preparing dual ligand co-functionalized fluorescent Au nanoclusters (NCs) by using threonine (Thr) and 11-mercaptoundecanoic acid (MUA) as assorted reductants and capping agents in aqueous solution at room temperature. Fluorescence spectra, high-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and infrared (IR) spectroscopy were performed to demonstrate the optical properties and chemical composition of the as-prepared AuNCs. They possess many attractive features such as near-infrared emission (λem = 606 nm), a large Stoke's shift (>300 nm), high colloidal stability (pH, temperature, salt, and time stability), and water dispersibility. Subsequently, the as-prepared AuNCs were used as a versatile probe for “turn off” sensing of Hg2+ based on aggregation-induced fluorescence quenching and for “turn-on” sensing of oxytetracycline (OTC). This assay provided good linearity ranging from 37.5 to 3750 nM for Hg2+ and from 0.375 to 12.5 μM for OTC, with detection limits of 8.6 nM and 0.15 μM, respectively. Moreover, the practical application of this assay was further validated by detecting OTC in human serum samples.
Keywords: Dual ligand co-functionalized; Gold nanoclusters; Fluorescence; Hg2+ ; Oxytetracycline

Lipidomic profiling of tryptophan hydroxylase 2 knockout mice reveals novel lipid biomarkers associated with serotonin deficiency by Rui Weng; Sensen Shen; Casey Burton; Li Yang; Honggang Nie; Yonglu Tian; Yu Bai; Huwei Liu (2963-2973).
Serotonin is an important neurotransmitter that regulates a wide range of physiological, neuropsychological, and behavioral processes. Consequently, serotonin deficiency is involved in a wide variety of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, schizophrenia, and depression. The pathophysiological mechanisms underlying serotonin deficiency, particularly from a lipidomics perspective, remain poorly understood. This study therefore aimed to identify novel lipid biomarkers associated with serotonin deficiency by lipidomic profiling of tryptophan hydroxylase 2 knockout (Tph2−/−) mice. Using a high-throughput normal-/reversed-phase two-dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry (NP/RP 2D LC–QToF-MS) method, 59 lipid biomarkers encompassing glycerophospholipids (glycerophosphocholines, lysoglycerophosphocholines, glycerophosphoethanolamines, lysoglycerophosphoethanolamines glycerophosphoinositols, and lysoglycerophosphoinositols), sphingolipids (sphingomyelins, ceramides, galactosylceramides, glucosylceramides, and lactosylceramides) and free fatty acids were identified. Systemic oxidative stress in the Tph2−/− mice was significantly elevated, and a corresponding mechanism that relates the lipidomic findings has been proposed. In summary, this work provides preliminary findings that lipid metabolism is implicated in serotonin deficiency.
Keywords: Serotonin deficiency; Tryptophan hydroxylase 2 knockout (Tph2−/−) mice; Lipidomics; Lipid biomarkers; Two-dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry (2D LC–QToF-MS)

PEPR: pipelines for evaluating prokaryotic references by Nathan D. Olson; Justin M. Zook; Daniel V. Samarov; Scott A. Jackson; Marc L. Salit (2975-2983).
The rapid adoption of microbial whole genome sequencing in public health, clinical testing, and forensic laboratories requires the use of validated measurement processes. Well-characterized, homogeneous, and stable microbial genomic reference materials can be used to evaluate measurement processes, improving confidence in microbial whole genome sequencing results. We have developed a reproducible and transparent bioinformatics tool, PEPR, Pipelines for Evaluating Prokaryotic References, for characterizing the reference genome of prokaryotic genomic materials. PEPR evaluates the quality, purity, and homogeneity of the reference material genome, and purity of the genomic material. The quality of the genome is evaluated using high coverage paired-end sequence data; coverage, paired-end read size and direction, as well as soft-clipping rates, are used to identify mis-assemblies. The homogeneity and purity of the material relative to the reference genome are characterized by comparing base calls from replicate datasets generated using multiple sequencing technologies. Genomic purity of the material is assessed by checking for DNA contaminants. We demonstrate the tool and its output using sequencing data while developing a Staphylococcus aureus candidate genomic reference material. PEPR is open source and available at https://github.com/usnistgov/pepr .
Keywords: Microbiology; Whole genome sequencing; Bioinformatics

Quantifying oxygen in paper-based cell cultures with luminescent thin film sensors by Matthew W. Boyce; Rachael M. Kenney; Andrew S. Truong; Matthew R. Lockett (2985-2992).
Paper-based scaffolds are an attractive material for generating 3D tissue-like cultures because paper is readily available and does not require specialized equipment to pattern, cut, or use. By controlling the exchange of fresh culture medium with the paper-based scaffolds, we can engineer diffusion-dominated environments similar to those found in spheroids or solid tumors. Oxygen tension directly regulates cellular phenotype and invasiveness through hypoxia-inducible transcription factors and also has chemotactic properties. To date, gradients of oxygen generated in the paper-based cultures have relied on cellular response-based readouts. In this work, we prepared a luminescent thin film capable of quantifying oxygen tensions in apposed cell-containing paper-based scaffolds. The oxygen sensors, which are polystyrene films containing a Pd(II) tetrakis(pentafluorophenyl)porphyrin dye, are photostable, stable in culture conditions, and not cytotoxic. They have a linear response for oxygen tensions ranging from 0 to 160 mmHg O2, and a Stern-Volmer constant (K sv) of 0.239 ± 0.003 mmHg O2 −1. We used these oxygen-sensing films to measure the spatial and temporal changes in oxygen tension for paper-based cultures containing a breast cancer line that was engineered to constitutively express a fluorescent protein. By acquiring images of the oxygen-sensing film and the fluorescently labeled cells, we were able to approximate the oxygen consumption rates of the cells in our cultures. Graphical Abstract Schematic of a paper-based culture seeded with fluorescent cells, and an oxygen-sensitive film
Keywords: Paper-based scaffold; 3D culture; Oxygen gradient; Oxygen sensor; Thin film; Fluorescence imaging

Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer’s disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall, we have demonstrated OxcysDML as a simple, rapid, robust, and inexpensive redox proteomic approach that is useful for gaining deeper insight into the proteome of AD. Graphical abstract OxcysDML enables the proteome comparision of cysteine reversible oxidations from two biological conditions
Keywords: OxcysDML; Cysteine; Dimethylation; Alzheimer’s disease; Stable isotope labeling; Mass spectrometry; Quantitative proteomics; Redox proteomics; Cysteine oxidation

Direct selective sensing of arginine in central nervous systems remains very essential to understanding of the molecular basis of some physiological events. This study presents the first demonstration on a simple yet effective method for arginine sensing with gold nanoparticles (Au-NPs) as the signal readout. The rationale for the method is based on the pH-dependent feature of the interionic interaction between cysteine and arginine. At pH 6.0, cysteine can only interact with arginine through the ion-pair interaction and such interaction can lead to the changes in both the solution color and UV-vis spectrum of the cysteine-protected Au-NPs upon the addition of arginine. These changes are further developed into an analytical strategy for effective sensing of arginine by rationally controlling the pH values of Au-NP dispersions with the ratio of the absorbance at 650 nm (A 650) to that at 520 nm (A 520) (A 650/A 520) as a parameter for analysis. The method is responsive to arginine without the interference from other species in the cerebral system; under the optimized conditions, the A 650/A 520 values are linear with the concentration of arginine within a concentration range from 0.80 to 64 μM, yet remain unchanged with the addition of other kinds of amino acids or the species in the central nervous system into the Au-NPs dispersion containing cysteine. The method demonstrated here is reliable and robust and could thus be used for detection of the increase of arginine in central nervous systems. Graphical Abstract A simple yet highly selective method for arginine sensing with gold nanoparticles (Au-NPs) as the signal readout was developed based on the interionic interaction between cysteine and arginine
Keywords: Arginine; Gold nanoparticles; Interionic interaction; In vivo analysis

Evaluation of a reconfigurable portable instrument for copper determination based on luminescent carbon dots by Alfonso Salinas-Castillo; Diego P. Morales; Alejandro Lapresta-Fernández; María Ariza-Avidad; Encarnación Castillo; Antonio Martínez-Olmos; Alberto J. Palma; Luis Fermin Capitan-Vallvey (3013-3020).
A portable reconfigurable platform for copper (Cu(II)) determination based on luminescent carbon dot (Cdots) quenching is described. The electronic setup consists of a light-emitting diode (LED) as the carbon dot optical exciter and a photodiode as a light-to-current converter integrated in the same instrument. Moreover, the overall analog conditioning is simply performed with one integrated solution, a field-programmable analog array (FPAA), which makes it possible to reconfigure the filter and gain stages in real time. This feature provides adaptability to use the platform as an analytical probe for carbon dots coming from different batches with some variations in luminescence characteristics. The calibration functions obtained that fit a modified Stern-Volmer equation were obtained using luminescence signals from Cdots quenching by Cu(II). The analytical applicability of the reconfigurable portable instrument for Cu(II) using Cdots has been successfully demonstrated in tap water analysis.
Keywords: Carbon dots; Luminescence; Reconfigurable electronics; FPAA; Copper determination

Hydroxyl radical protein footprinting coupled with mass spectrometry has become an invaluable technique for protein structural characterization. In this method, hydroxyl radicals react with solvent exposed amino acid side chains producing stable, covalently attached labels. Although this technique yields beneficial information, the extensive list of known oxidation products produced make the identification and quantitation process considerably complex. Currently, the methods available for analysis either involve manual analysis steps, or limit the amount of searchable modifications or the size of sequence database. This creates a bottleneck which can result in a long and arduous analysis process, which is further compounded in a complex sample. Here, we report the use of a new footprinting analysis method for both peptide and residue-level analysis, demonstrated on the GCaMP2 synthetic construct in calcium free and calcium bound states. This method utilizes a customized multi-search node workflow developed for an on-market search platform in conjunction with a quantitation platform developed using a free Excel add-in. Moreover, the method expedites the analysis process, requiring only two post-search hours to complete quantitation, regardless of the size of the experiment or the sample complexity. Graphical Abstract Process overview of Proteome Discoverer data analysis strategy
Keywords: Oxidative modification; Mass spectrometry; Data analysis; Protein footprinting

Development of magnetic molecularly imprinted polymers for selective extraction: determination of citrinin in rice samples by liquid chromatography with UV diode array detection by Javier L. Urraca; José F. Huertas-Pérez; Guillermo Aragoneses Cazorla; Jesus Gracia-Mora; Ana M. García-Campaña; María Cruz Moreno-Bondi (3033-3042).
In this work, we report the synthesis of novel magnetic molecularly imprinted polymers (m-MIPs) and their application to the selective extraction of the mycotoxin citrinin (CIT) from food samples. The polymers were prepared by surface imprinting of Fe3O4 nanoparticles, using 2-naphtholic acid (2-NA) as template molecule, N-3,5-bis(trifluoromethyl)phenyl-N'-4-vinylphenyl urea and methacrylamide as functional monomers and ethyleneglycol dimethacrylate as cross-linker. The resulting material was characterized by transmission electron microscopy (TEM), and X-ray diffraction (XRD) and Fourier transform infrared spectroscopies (FT-IR). The polymers were used to develop a solid-phase extraction method (m-MISPE) for the selective recovery of CIT from rice extracts prior to its determination by HPLC with UV diode array detection. The method involves ultrasound-assisted extraction of the mycotoxin from rice samples with (7:3, v/v) methanol/water, followed by sample cleanup and preconcentration with m-MIP. The extraction (washing and elution) conditions were optimized and their optimal values found to provide CIT recoveries of 94–98 % with relative standard deviations (RSD) less than 3.4 % (n = 3) for preconcentrated sample extracts (5 mL) fortified with the analyte at concentrations over the range 25–100 μg kg−1. Based on the results, the application of the m-MIPs facilitates the accurate and efficient determination of CIT in rice extracts. Graphical Abstract Novel magnetic molecularly imprinted polymers (m-MIPs) for citrinin (CIT) have been obtained and applied to the selective extraction of the mycotoxin from rice samples
Keywords: Citrinin; Mycotoxins; Molecular imprinting; Cleanup; Rice