Phosphomolybdenum Blue Detection – A Review of Characteristics, Achievements, Challenges and Future Prospects

Phosphate detection in the environment (especially, water bodies) is very essential in view of its consequent pollution effects on eutrophication. Continuous monitoring of phosphate concentration (and phosphorus compounds in general) in water samples has been based on phosphomolybdenum blue formation coupled with spectrophotometric detection. Continu­ous flow injection analyses (FIA) are well known to present numerous advantages over batch methods. Furthermore, the development of on-chip micro-channel analytical (μFIA) systems begun and have gained much attention within the last two decades in view of the striking advantages over conventional FIA techniques. This paper reviews published information on detection of phosphomolybdenum blue in conventional systems as well as on micro-chip. It reports on the challenges encountered (interference from other complex anions), the achieve­ments made so far (intereference removal by electrokinetic separation) and what the future holds (simultaneous determination)

Seismic chimney characterisation in the North Sea – Implications for pockmark formation and shallow gas migration

Fluid-escape structures within sedimentary basins permit pressure-driven focused fluid flow through inter-connected faults, fractures and sediment. Seismically-imaged chimneys are recognised as fluid migration pathways which cross-cut overburden stratigraphy, hydraulically connecting deeper strata with the seafloor. However, the geological processes in the sedimentary overburden which control the mechanisms of genesis and temporal evolution require improved understanding. We integrate high resolution 2D and 3D seismic reflection data with sediment core data to characterise a natural, active site of seafloor methane venting in the UK North Sea and Witch Ground Basin, the Scanner pockmark complex. A regional assessment of shallow gas distribution presents direct evidence of active and palaeo-fluid migration pathways which terminate at the seabed pockmarks. We show that these pockmarks are fed from a methane gas reservoir located at 70 metres below the seafloor. We find that the shallow reservoir is a glacial outwash fan, that is laterally sealed by glacial tunnel valleys. Overpressure generation leading to chimney and pockmark genesis is directly controlled by the shallow geological and glaciogenic setting. Once formed, pockmarks act as drainage cells for the underlying gas accumulations. Fluid flow occurs through gas chimneys, comprised of a sub-vertical gas-filled fracture zone. Our findings provide an improved understanding of focused fluid flow and pockmark formation within the sediment overburden, which can be applied to subsurface geohazard assessment and geological storage of CO2

Direct Quantitative Analysis of Multiple Micrornas

MicroRNAs (miRNAs) play a significant role in gene regulation and have been shown to be deregulated in various diseases. Specific sets of deregulated miRNAs, termed miRNA fingerprints, can distinguish diseased from healthy samples. Detecting disease-specific fingerprints could be used in diagnostics, and there are significant efforts toward developing miRNA-detection methods for this purpose. In my project, I developed the first direct quantitative analysis of multiple miRNAs (DQAMmiR) which does not require any modifications to the target miRNAs. DQAMmiR is a hybridization assay which utilizes the separative abilities of capillary electrophoresis to analyze multiple miRNAs. I used two separation-enhancement approaches: 1) drag tags on the DNA probes to separate multiple hybrids and 2) single-strand DNA binding protein (SSB) in the run buffer to separate excess, unbound DNA probes from the hybrids. In the proof-of-principle work, I detected three miRNAs directly from a cell lysate with a limit of detection of 100 pM. I was able to further increase the number of detectable miRNAs by conjugating short peptides of varying length to the DNA probes. These peptides acted as drag tags which allowed for the separation and detection of 5 miRNAs. To lower the limit of detection I combined DQAMmiR with isotachophoresis, an in-capillary pre-concentration technique. The limit of detection improved by two orders of magnitude (from 100 pM down to 1 pM), allowing the detection of low abundance miRNAs. To improve the specificity of DQAMmiR I incorporated locked nucleic acid (LNA) bases into the probes to normalize the melting temperature of all target miRNA hybrids. This allowed me to use a single hybridization temperature, at which all target miRNA hybrids remained intact while single-nucleotide mismatches melted. Also, a dual capillary temperature technique was developed in which separation started with a high capillary temperature, required for proper hybridization, and continued at a low capillary temperature required for quality electrophoretic separation of the hybrids. I was able to combine all of these improvements to DQAMmiR while using an automated, commercially available instrument, making it an accurate, quantitative, specific, sensitive, time and cost efficient method for the analysis of multiple miRNAs

Influence of Magnetic Nanoparticles and Magnetic Stress on an Ionic Liquid Electrospray Source

Two electrospray sources were developed to operate on an ionic liquid ferrofluid; one source was a pressure‑fed capillary electrospray source and the other was a novel electrospray source which used a magnetically‑induced instability to produce a peak from which an electric field could extract electrospray. Multiple characteristics of electrospray operation were examined for both sources using faraday plates/cups, a quartz crystal microbalance, a retarding potential analyzer, and a time-of-flight mass spectrometer. The ILFF electrosprays for a capillary source were shown to operate in a mixed ion/droplet regime. The mass flow of the electrospray beam was primarily transported by larger particles (potential droplets) within it. The magnetic nanoparticles increased the required flowrate and extraction potential of the source, as well as the emission current at a given flowrate. The nanoparticles also influenced the beam divergence and energy of an electrospray, increasing and decreasing each respectively with higher concentrations of NPs. The magnetic field had significant influence on the required flowrate of the electrospray, as it reduced the minimum stable flowrate by upwards of 16 percent. It also was shown to decreased the emission current of ILFF electrosprays for a given flowrate, while concurrently increasing the beam energy of particles in the electrospray. Other effects of magnetic field on electrospray characteristics were either inconclusive or insignificant

A review

Funding Information: PhD grant PD/BDE/150627/2020 was financed by Fundação para a Ciência e Tecnologia (FCT - Portugal) and Volkswagen Autoeuropa. Funding Information: The authors would like to thank Fundação para a Ciência e Tecnologia (FCT - Portugal), and Volkswagen Autoeuropa for co-financing the doctoral grant PD/BDE/150627/2020. Publisher Copyright: © 2023 The AuthorsIon Mobility Spectrometry (IMS) has gained relevance in the field of analytical techniques over the past decades. If compared with well-established techniques like mass spectrometry or infrared spectroscopy, IMS is considerably less developed or employed in specific fields but presents promising results and a substantial margin for improvements. Its outstanding sensitivity and selectivity, analytical flexibility, instrumental versatility and almost real-time results capacity have contributed to elevate IMS among the main analytical techniques for the detection of volatile organic compounds. Due to its growth potential, it is mandatory to assess in which scientific fields IMS has played a relevant role in the past years of academic research and understand in which areas it can become equally important in the near future. For this purpose, hundreds of scientific works from the past ten years were addressed and the most relevant were reviewed in this work. Three main categories of IMS applications were defined to group the reviewed articles: Environmental and Safety Research, Health Research and Food Research. In addition, some original studies were specifically developed for this review paper, to act as elucidative examples. The working principle of the IMS is included for clarification purposes. A glossary of all the mentioned compounds was also included. Throughout the text, it is clear how relevant IMS has become and how diverse its applicability can be, ranging from simpler topics like fraud detection to more complex ones like pathologies diagnosis. It is safe to say that IMS has been, step by step, gaining relevance as an analytical technique and its potential for supporting many diverse scientific fields is evident.publishersversionpublishe

Analysis and Fingerprinting of Glycosaminoglycans

Heparin is a complex mixture of sulfated polysaccharides derived from animals and one of the oldest drugs in use. While an efficacious anticoagulant, heparin is beset by side effects and pharmacokinetic difficulties. Low molecular weight heparins (LMWH) are made by depolymerizing unfractionated heparin (UFH) and present improvements in these areas. However, they still retain a phenomenally high level of complexity due to their polydispersity and the introduction of non-native structural features. This makes the structural characterization LMWHs a daunting task. This work details the development of a novel capillary electrophoretic (CE) method for fingerprinting LMWHs. Since their complexity normally results in a nearly featureless electropherogram, polyalkylamines were used as a resolving agents to yield highly resolved and reproducible fingerprints characteristic of the LMWH being investigated. Linear polyamines of resolved LMWH in a manner dependent on chain length and charge density, while cyclic polyamines were incapable of resolution. Longer length glycosaminoglycans such as UFH and chondroitin sulfate were not successfully fingerprinted as they lacked run to run consistency. Further investigation into the mode of polyamine binding showed that they bound to LMWH via a two site binding model, indicating the presence of specific sites on LMWH that tightly bind polyamines. Upon the saturation of these sites, the polyamines continue to interact via general electrostatic binding. Pentaethylenehexamine was also able to separate the known contaminant oversulfated chondroitin sulfate from UFH. In July of 2010, the US food and drug administration approved a generic for the widely used LMWH enoxaparin, a questionable move due to the difficulties of proving the equivalence of such a complex mixture. A comparison of the brand and generic batches of enoxaparin using the fingerprinting method revealed striking similarities, bolstering the generic’s claim of equivalency and providing a protocol for the evaluation of other biosimilar LMWHs. This is the first work utilizing CE in developing high resolution fingerprints of LMWH. It presents a noteworthy method for quality assessment of LMWH and provides the basis for designing other small molecule probes for the analysis of complex glycosaminoglycans

Chemical Characterization of Wild Maine Cranberries

This study was conducted to analyze wild Maine cranberries for anthocyanin (ACY), anthocyanidin, organic acid, L-ascorbic acid, and moisture content. Cranberries were analyzed in both the fiesh and fiozen states. Analysis for ACY and anthocyanidin content was performed by High Performance Liquid Chromatography (HPLC) and Capillary Electrophoresis (CE); organic acids by HPLC; and L-ascorbic acid (L-AA) and moisture by AOAC methods for titration and drying, respectively. ACY results showed similar qualitative profiles for all cultivars except one where no detectable ACY were present. Four major peaks and two minor peaks were detected, which have been tentatively identified as the galactosides (Gal), arabinosides (Arab), and glucosides (Glu) of cyanidin (Cy) and peonidin (Pn). Quantitatively, significant differences (p\u3c0.05) existed among the different cultivars for total and individual ACY content, with totals ranging fiom 9.64 to 126.05 mgA00g and 13.1 1 to 94.48 mg/100g for fiesh and fiozen samples, respectively (results reported as Cy-3-Gal). The proportion of individual ACY content varied notably with Cy-3-Gal comprising 20-35% of the total, Cy-3-Arab 14-22%, Pn-3-Gal30-45%, and Pn-3-Arab 13-18%. Aglycones obtained through acid hydrolysis of the ACY and analyzed by HPLC resulted in 55% Cy and 35% Pn. The CE method developed in this study employed an acidic run buffer, which kept the aglycones in their flavyliurn cation form. Thus, the same concentrations of pigments used in HPLC were applied to CE. For comparison of methods, HPLC and CE analyses were performed on commercial cranberries; Results showed that the methods were comparable despite the noisy baseline of the CE online detection. The correlation coefficient was 0.967. The primary organic acids in cranberries are citric, malic, quinic, and L-ascorbic. Mean organic acid content of fresh and frozen berries varied slightly (2041 mg/100g and 2035 mg/100g, respectively) despite the large differences in individual acids. Malic was the primary acid in fresh berries at 64%. Citric and quinic made up 25% and 11%, respectively. In the frozen berry, citric and malic acids were evenly matched at 42% and 4 1 %, respectively. Quinic remained low at 17%

Deep Sequencing of the Small RNAs Derived from Two Symptomatic Variants of a Chloroplastic Viroid: Implications for Their Genesis and for Pathogenesis

Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21–24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations. To overcome this restraint we have examined by deep sequencing (Solexa-Illumina) and computational approaches the vd-sRNAs accumulating in GF-305 peach seedlings infected by two molecular variants of Peach latent mosaic viroid (PLMVd) inciting peach calico (albinism) and peach mosaic. Our results show in both samples multiple PLMVd-sRNAs, with prevalent 21-nt (+) and (−) RNAs presenting a biased distribution of their 5′ nucleotide, and adopting a hotspot profile along the genomic (+) and (−) RNAs. Dicer-like 4 and 2 (DCL4 and DCL2, respectively), which act hierarchically in antiviral defense, likely also mediate the genesis of the 21- and 22-nt PLMVd-sRNAs. More specifically, because PLMVd replicates in plastids wherein RNA silencing has not been reported, DCL4 and DCL2 should dice the PLMVd genomic RNAs during their cytoplasmic movement or the PLMVd-dsRNAs generated by a cytoplasmic RNA-dependent RNA polymerase (RDR), like RDR6, acting in concert with DCL4 processing. Furthermore, given that vd-sRNAs derived from the 12–14-nt insertion containing the pathogenicity determinant of peach calico are underrepresented, it is unlikely that symptoms may result from the accidental targeting of host mRNAs by vd-sRNAs from this determinant guiding the RNA silencing machinery

Maskless additive manufacturing of micro structures by laser sintering of nanoparticles

The focus of this study was to investigate an alternate microfabrication process of sintering nanoparticles using a patterned laser light to generate two dimensional (2D) and three dimensional (3D) geometries. With trends of producing electronic and medical devices using cheaper and faster technologies, a new procedure was developed to deposit and bond miniature patterns of silver nanoparticles onto silicon substrates. The process consists of spraying silver nanoparticles onto the substrate while a patterned or modulated laser beam is focused onto the sprayed nanoparticles. These nanoparticles exposed under the modulated laser light undergo partial melting or sintering and adhere to the substrate. The substrate is then cleaned to remove the unexposed nanoparticles from the surface. The deposition thus obtained from the process yields a 2D micropattern. 3D parts are produced using a layer by layer manufacturing process in which a two dimensional pattern is deposited over another pattern repeatedly until the desired elevation is obtained. The initial part of the thesis will discuss the experimental investigation on the influence of process parameter (i.e. flowrate, electric field distance, standoff distance and extractor size) on controlling the length, size and distribution of the spray to deposit nanoparticles. Later, the results of deposition and fabrication of microstructures using the process are presented --Abstract, page iii

Mass spectrometry-based applications and analytical method development for metabolomics

Metabolites are small molecules present in a biological system that have multiple important biological functions. Changes in metabolite levels reflect genetic and environmental alterations and play a role in multiple diseases. Metabolomics is a discipline that aims to analyze all the small molecules in a biological system simultaneously. Since metabolites represent a diverse group of compounds with varying chemical and physical properties with a wide concentration range, metabolomic analysis is technically challenging. Due to its high sensitivity and selectivity, mass spectrometry coupled with chromatographic separation is the most commonly used analytical tool. Currently, there is no comprehensive universal analytical tool to detect all metabolites simultaneously and multiple methods are required. The aim of this study was to develop and apply mass spectrometry-based analytical methods for metabolomics studies. Neonatal rodents can fully regenerate their hearts after an injury. However, this regenerative capacity is lost within 7 days after birth. The molecular mechanism behind this phenomenon is unknown and understanding the biology behind this loss of regeneration capacity is necessary for the development of regeneration-inducing therapies. To investigate this mechanism, changes in mouse heart metabolite, protein, and transcript levels during the early postnatal period were studied. Non-targeted metabolomics methods utilizing liquid chromatography-mass spectrometry (LC-MS) and two-dimensional gas chromatography-mass spectrometry (GCxGC-MS) were applied to detect the metabolic changes of neonatal mouse hearts. Two complementary techniques increased metabolite coverage. A total of 151 identified metabolites showed differences in the neonatal period, reflecting changes in multiple metabolic pathways. The most significant changes observed in all levels (metabolite, protein, and transcript) were branched chain amino acid (BCAA) catabolism, fatty acid metabolism, and the mevalonate and ketogenesis pathways, thus revealing possible associations with regeneration capacity or regulation of the cardiomyocyte cell cycle. Insulin resistance (IR), metabolic syndrome, and type 2 diabetes have been shown to induce metabolic changes; the origin of the changes is unknown. In this study, human serum metabolite profiles from non-diabetic individuals were associated with IR. Gut microbiota were identified as a possible origin of the metabolic changes. Serum metabolites were detected with GCxGC-MS and lipids with LC-MS method. In total, 19 serum metabolite clusters were significantly associated with the IR phenotype, including 26 polar metabolites from five separate clusters and 367 lipids from 14 clusters. IR and changed metabolites were further associated with gut microbiota metagenomics and gut microbiota functional modules, showing that gut microbiota impacts the human serum metabolites associated with IR. Individuals with the IR phenotype had increased BCAA levels, which was influenced by bacterial species with increased BCAA biosynthesis potential and the absence of species with active bacterial inward BCAA transport. Sample throughput is often limited when chromatographic separation is used in metabolomics applications; a short analysis time is of great importance in large metabolic studies. The feasibility of direct infusion electrospray microchip MS (chip-MS) for global non-targeted metabolomics to detect metabolic differences between two cell types was studied and was compared to the more traditional LC-MS method. We observed that chip-MS was a rapid and simple method that allowed high sample throughput from small sample volumes. The chip-MS method was capable of separating cells based on their metabolic profiles and could detect changes of several metabolites. However, the selectivity of chip-MS was limited compared to LC-MS and chip-MS suffers more from ion suppression. Many biologically important low-abundance metabolites are not detectable with non-targeted metabolomics methods and separate more sensitive targeted methods are required. An in-house developed capillary photoionization (CPI) source was shown to have high ion transmission efficacy and high sensitivity towards non-polar compounds such as steroids. In this study, the CPI prototype was developed to increase its sensitivity. The feasibility of the ion source for the quantitative analysis of biological samples was studied by analyzing 18 endogenous steroids in urine with gas chromatography capillary photoionization tandem mass spectrometry (GC-CPI-MS/MS). The GC-CPI-MS/MS method showed good chromatographic resolution, acceptable linearity and repeatability, and low limits of detection (2-100 pg mL-1). In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the human urine samples. Additionally, the applicability of the CPI interface for LC applications was explored for the first time using low flow rates. The feasibility of the LC-CPI-MS/MS for the quantitative analysis of four steroids was studied in terms of linearity, repeatability, and limits of detection. The method showed good quantitative performance and high sensitivity at a low femtomole level.Metaboliitit ovat molekyylipainoltaan pieniä aineenvaihduntatuotteita, joilla on merkittäviä biologisia tehtäviä elimistössä. Aineenvaihduntatuotteiden muutoksien on osoitettu liittyvän moniin sairauksiin, jonka takia niiden analysoiminen on tärkeää sairauksien diagnosoimiseksi, ennustamiseksi, sairausmekanismin selvittämiseksi, sekä lääkehoidon kehittämiseksi. Metabolomiikka pyrkii analysoimaan aineenvaihduntatuotteet samanaikaisesti biologisista näytteistä ja vertailemaan aineenvaihduntatuotteiden tasoja eri fysiologisten tilojen välillä ja sitä voidaankin hyödyntää useissa lääketieteellisen tutkimuksen eri vaiheissa. Aineenvaihduntatuotteiden pitoisuudet ja kemialliset ominaisuudet kuitenkin vaihtelevat merkittävästi, mikä tekee aineenvaihduntatuotteiden samanaikaisen analysoinnin haastavaksi. Tällä hetkellä ei ole olemassa yhtä kaikkien aineenvaihduntatuotteiden samanaikaiseen analyysiin soveltuvaa menetelmää. Parhaiten metaboliittien analyysiin soveltuu massaspektrometria, usein yhdistettynä kromatografisiin erotustekniikoihin, sen ylivoimaisen herkkyyden ja spesifisyyden vuoksi. Tässä työssä pyrittiin soveltamaan massaspektrometrisiä kohdentamattomia profilointimenetelmiä uuden biologisen tiedon tuottamiseksi muuttuneista aineenvaihduntatuotteista. Työssä pyrittiin myös kehittämään uusia herkempiä ja nopeampia massaspektrometrisiä menetelmiä metabolomiikkaan. Ensimmäisessä osatutkimuksessa kohdentamatonta metabolomiikkaa sovellettiin lääkehoidon kohdemolekyylin etsintään solujen uusiutumisen aktivoimiseksi massiivisia soluvaurioita aiheuttavien sairauksien hoidossa. Aineenvaihduntareiteissä tapahtuvia muutoksia tutkittiin hiirien sydämistä, jotka menettävät sydänsolujen uusiutumiskyvyn pian syntymän jälkeen. Metabolomiikalla havaittiin muutoksia useilla aineenvaihduntareiteillä syntymän jälkeisellä ajanjaksolla, jolloin sydänsolujen uusiutumiskapasiteetti heikkenee. Tuloksia yhdistettiin muiden biomolekyylien analyysituloksiin ja saatiin kattavaa tietoa syntymän jälkeisistä molekyylimuutoksista. Erityisesti ketogeneesi-aineenvaihduntareitin havaittiin vaikuttavan solujen jakautumiseen ja lisääntymiseen. Insuliiniresistenssin, metabolisen oireyhtymän ja tyypin 2 diabeteksen on osoitettu aiheuttavan metabolisia muutoksia ihmisissä, mutta muutosten syy on ollut tuntematon. Työn toisessa osatutkimuksessa tutkittiin suoliston mikrobiston yhteyttä insuliiniresistenssiin ja metabolisiin muutoksiin seerumissa. Tutkimuksessa havaittiin seerumin metaboliittien muutoksilla olevan yhteys insuliiniresistenssiin, sekä yksilöiden suolistomikrobeihin ja niiden toimintoihin. Insuliiniresistentillä ryhmällä havaittiin kohonneet haaroittuneiden aminohappojen pitoisuudet seerumissa, sekä enemmän bakteerilajeja, jotka syntetisoivat haaroittuneita aminohappoja. Tutkimus antoi viitteitä, että suolistomikrobeilla on yhteys insuliiniresistenssin ja tyypin 2 diabeteksen puhkeamiseen. Kolmannessa osatutkimuksessa tutkittiin suorasyöttömikrosirun soveltuvuutta kohdentamattomaan metaboliseen profilointiin vertailemalla menetelmää yleisesti käytettyyn nestekromatografia-massaspektrometria menetelmään solujen metaboliittien erojen havaitsemiseksi. Mikrosiru-menetelmän etuna on nopea alle minuutin analyysiaika, joka mahdollistaa ison näytemäärän analysoinnin lyhyessä ajassa pienestä näytemäärästä. Erot solujen metabolisten profiileiden välillä pystyttiin havaitsemaan molemmilla menetelmillä. Mikrosiru-menetelmän selektiivisyys oli kuitenkin heikompi ja ionien supressio massaspektrometrissa oli merkittävämpää. Neljännessä ja viidennessä osatutkimuksessa tutkittiin kapillaarifotoionisaatio ionisaatiotekniikan soveltuvuutta metabolomiikkaan. Kohdentamattomien profilointimenetelmien herkkyys ei riitä matalien pitoisuuksien analysointiin, jonka vuoksi myös kohdennettuja herkempiä menetelmiä tarvitaan tiettyjen yhdisteryhmien määrittämiseksi. Kapillaarifotoionisaation soveltuvuutta tutkittiin kehittämällä menetelmä steroidien määrittämiseksi virtsasta kaasukromatografia-massaspektrometrilla. Menetelmällä saavutettiin hyvä herkkyys ja menetelmän todettiin soveltuvan steroidien kvantitatiiviseen analytiikkaan biologisesta matriisista. Kapillaarifotoionisaation soveltuvuutta steroidien analysoimiseksi myös nestekromatografia-massaspektrometrialla tutkittiin ensimmäistä kertaa. Tutkimuksessa havaittiin kapillaarifotoionisaation soveltuvan myös nestekromatografiaan pienillä virtausnopeuksilla. Kohdentamattomalla metabolomiikalla saavutettiin merkittävää tietoa muuttuneista aineenvaihduntatuotteista ja havaittavien metaboliittien määrää laajennettiin käyttämällä kahta rinnakkaista menetelmää. Työssä kapillaarifotoionisaation ja mikrosirutekniikan osoitettiin soveltuvan käytettäväksi metabolomiikassa