Laser-induced Fluorescence Detection Research Articles

Kinetic Study of the Reactions of AlO with H2O and H2; Precursors to Stellar Dust Formation

AlO is relatively abundant around oxygen-rich Asymptotic Giant Branch (AGB) stars, where it can react with major gas-phase species such as H2 and H2O to form AlOH. These Al-containing species are the likely precursors of refractory alumina nanoparticles, which may provide the nuclei for dust formation. In the present study, the kinetics of these AlO reactions were measured from 295 to 780 K using the pulsed laser photolysis of Al(C5H7O2)3, with time-resolved laser induced fluorescence detection of AlO. The experimental results were interpreted using quantum chemistry calculations and a Master Equation solver for reactions with multiple energy wells. For the recombination reaction AlO + H2O (+ N2) → Al(OH)2, log10(krec,0/cm6 molecule–2 s–1) = −32.9185 + 8.80276 log10(T) – 2.4723(log10(T))2; log10(krec,∞/cm3 molecule–1 s–1) = −19.4686 + 7.62037 log10(T) – 1.47214(log10(T))2; Fc = 0.28 (uncertainty ±13% from 295 to 760 K). For the bimolecular reactions, k(AlO + H2O → AlOH + OH) = (3.89 ± 0.47) × 10–10 exp(−(1295 ± 150)/T) and k(AlO + H2 → AlOH + H) = (5.37 ± 0.52) × 10–13 (T/300)(2.77 ± 0.19) exp(−(2190 ± 110)/T) cm3 molecule–1 s–1. Rate coefficients for Al + H2O → AlOH + H, AlOH + H → AlO + H2 or Al + H2O, and the absorption cross sections of AlOH and AlO were calculated theoretically. Al chemistry around an O-rich AGB star was then investigated using a β-trajectory model, which predicts that AlOH is the major gas-phase Al species beyond two stellar radii and shows that the relative AlO abundance is very sensitive to the AlOH photolysis rate.

Take a good catch at the scat: carboxylic and sulfonic acid profiles as a non-invasive tool for species identification and sex determination in neotropical carnivores

ABSTRACT There is an increasing interest in the use of noninvasive monitoring methods, such as the use of chemical profiles obtained from noninvasive samples. Here, we propose the use of carboxylic and sulfonic acids profiles obtained from scats as a noninvasive tool to identify mammalian carnivoran species and for sex determination. We used High-Performance Liquid Chromatography (HPLC) coupled to a Laser-Induced Fluorescence (LIF) detection system (HPLC-LIF) to retrieve carboxylic and sulfonic acids profiles from scats of five mammalian carnivores: Puma concolor, Herpailurus yagouaroundi, Leopardus pardalis and Felis catus (all Felidae) and Nasua nasua (Procyonidae). We were able to unambiguously identify the species and determinate the sex for all fecal samples through the pattern of presence-absence of carboxylic and sulfonic acids (digital coding). The method allowed the discrimination of the evaluated species of the Carnivora, even between closely related and ecologically similar felids. Moreover, our method is low cost and can be executed in a short period of time. Further investigation is necessary to evaluate if the method’s discriminatory power is applicable to other mammalian species. Additionally, the method’s potential to ascertain other intraspecific traits such as age, identity, reproductive and health status is yet to be determined.

Simple, rapid, and reproducible capillary gel electrophoresis separation and laser-induced fluorescence detection of DNA topoisomers with unmodified fused silica separation capillaries.

The topology of DNA is a critical quality attribute for plasmid-based pharmaceuticals, making quantification of trace levels of plasmid topoisomers an important analytical priority. An automated and cost-effective method based on capillary gel electrophoresis laser-induced fluorescence detection is described. The method outlined in this report is significant because it is easily implemented by any laboratory for which routine analyses of plasmid topology are critical for the development of new plasmid-based therapies as well as for quality control of gene therapies utilizing supercoiled DNA. Detection of topoisomers was achieved by incorporating ethidium bromide in the separation medium. The detector response was improved by 3 orders of magnitude by utilizing a 605-nm optical filter with a 15-nm bandwidth. Separations of linear, open circle, supercoiled, and multimer DNAplasmids ranging from 4.2 to 10.5kbp were accomplished in under 6min using an unmodified fused silica capillary (50-μm internal diameter). The background electrolyte was comprised of 0.5% gel, which was hydroxypropylmethyl cellulose, 1mM ethylenediaminetetraacetic acid, and 50mMN-(2-acetamido)-2-aminoethanesulfonic acid (pH of 6.25). The separations, which balanced the bulk electroosmotic flow, the electrophoretic mobility of the DNA, and gel sieving were dependent upon the pH of the electrolyte and the gel concentration. Reproducibility was dependent upon the procedure used to prepare the gel as well as other factors including the ethidium bromide concentration and capillary conditioning. A single unmodified capillary operated for more than 150 runs had an across-day migration time precision of 1% relative standard deviation and percent area precision of 10% relative standard deviation.

N-Glycosylation Profiling of Human Blood in Type 2 Diabetes by Capillary Electrophoresis: A Preliminary Study.

Currently, diagnosing type 2 diabetes (T2D) is a great challenge. Thus, there is a need to find rapid, simple, and reliable analytical methods that can detect the disease at an early stage. The aim of this work was to shed light on the importance of sample collection options, sample preparation conditions, and the applied capillary electrophoresis bioanalytical technique, for a high-resolution determination of the N-glycan profile in human blood samples of patients with type 2 diabetes (T2D). To achieve the profile information of these complex oligosaccharides, linked by asparagine to hIgG in the blood, the glycoproteins of the samples needed to be cleaved, labelled, and purified with sufficient yield and selectivity. The resulting samples were analyzed by capillary electrophoresis, with laser-induced fluorescence detection. After separation parameter optimization, the capillary electrophoresis technique was implemented for efficient N-glycan profiling of whole blood samples from the diabetic patients. Our results revealed that there were subtle differences between the N-glycan profiles of the diabetic and control samples; in particular, two N-glycan structures were identified as potential glycobiomarkers that could reveal significant changes between the untreated/treated type 2 diabetic and control samples. By analyzing the resulting oligosaccharide profiles, clinically relevant information was obtained, revealing the differences between the untreated and HMG-CoA reductase-inhibitor-treated diabetic patients on changes in the N-glycan profile in the blood. In addition, the information from specific IgG N-glycosylation profiles in T2D could shed light on underlying inflammatory pathophysiological processes and lead to drug targets.

Analysis of cyanide using fluorogenic derivatization and capillary electrophoresis

Samples containing cyanide were incubated at 85 °C in the presence of the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and glutamic acid, and analyzed by capillary electrophoresis utilizing post-separation laser-induced fluorescence detection in a sheath flow cuvette. The separation time on a 25 cm long capillary at 800 Vcm−1 was 3 min with the fluorescent product eluting at 107 s. Flushing of the capillary was not required between runs. Signal was proportional with cyanide concentration from 50 nM to 1.5 μM. LOD and LOQ were determined to be 26 and 87 nM respectively. As an application, free cyanide in five individual apple seeds was measured and found to range from 12 to 86 ng/mg, with a mean of 55 ± 32 ng/mg. As a means for the detection of amygdalin, cyanide was enzymatically produced from amygdalin using the enzymes β-glucosidase and mandelonitrile lyase. The cyanide was then reacted with FQ and injected onto the capillary. Amygdalin was detected at a concentration of 1 μM.

GlycoStore: A Platform for H/UPLC and Capillary Electrophoresis Glycan Data.

GlycoStore ( http://www.glycostore.org ) is an open access chromatographic and electrophoretic retention database of glycans characterized from glycoproteins, glycolipids, and biotherapeutics. It is a continuation of the GlycoBase project (Oxford Glycobiology Institute and National Institute for Bioprocessing Research and Training, Ireland) but addresses many of the technological limitations that impacted the growth of GlycoBase, in particular, improvements to the bioinformatics architecture, enhancing data annotations and coverage, and improving connectivity with external resources. The first release of GlycoStore (October 2017) contains over 850 glycan entries accompanied by 8500+ retention positions including data from: (1) fluorescently labelled released glycans determined using hydrophilic interaction chromatography (HILIC) ultrahigh-performance liquid chromatography (U/HPLC) and reversed phase (RP)-U/HPLC; (2) porous graphitized carbon chromatography (PGC) interfaced with ESI-MS/MS; and (3) capillary electrophoresis with laser induced fluorescence detection (CE-LIF). In this chapter, we outline the objectives of GlycoStore, and describe a selection of step-by-step workflows for navigating and browsing the information available. We also provide a short description of informatics tools available to query the database using Semantic technologies. The information presented in this chapter supplements our documentation knowledge base that describes interface improvements, new features and tools, and content updates ( https://unicarbkb.freshdesk.com/ ).

Rapid Antibody Glycoengineering in CHO Cells Via RNA Interference and CGE-LIF N-Glycomics.

The impact of the glycan distribution on the in vivo function and half-life of monoclonal antibodies has long motivated the genetic engineering of producer cells to achieve structures that enhance efficacy, safety and stability. To facilitate glycoengineeringof IgG-producing Chinese hamster ovary cells, we present a rapid protocol that involves the use of RNA interference for the knockdown of genes of interest coupled with capillary gel electrophoresis and laser-induced fluorescence detection (CGE-LIF) for fast, high-throughput glycan analysis. We apply this methodology to the Fut8 gene, responsible for the addition of core fucose, which is a typical target for increasing antibody-dependent cellular cytotoxicity.

Current State of Laser-Induced Fluorescence Spectroscopy for Designing Biochemical Sensors

Laser-induced fluorescence (LIF) has been a valuable analytical technique since the 1970s that has only been made more useful through advances in other scientific fields such as biochemistry. Moreover, advances in laser and detector technology have seen a decrease in LIF detector costs and an increase in their ease of use. These changes have allowed for LIF technology to be widely adopted for various sensor designs in combination with advanced instruments. With advances in biochemistry necessitating the detection of complex metabolites, labelling with fluorescent chemical reagents may be necessary to improve detection sensitivity. Furthermore, advances made in fluorescent labeling technologies have allowed for the use of LIF in the detection of nanoparticles as well as for imaging techniques using nanoparticles as signal amplifiers. This technology has become invaluable in the detection of environmental pollutants, monitoring of biological metabolites, biological imaging, and cancer diagnosis, making it one of the most valuable analytical science techniques currently available.

Open-source lab hardware: Low noise adjustable two-stage gain transimpedance amplifier with DC offset for low-light detection

An open-source, low noise, low cost, and tunable transimpedance amplifier is presented. The compact circuit board requires few parts and costs less than $65 USD. The transimpedance amplifier is intended for low-light detection and operation with commercial photomultiplier tubes (PMTs). It provides a much more cost-effective acquisition tool compared to competitive products on the market. The system can easily be assembled and modified to suit specific current sensing applications. The amplifier features two variable gains and an adjustable DC offset to optimize dynamic range and suppress potential bias in the signal. With a target bandwidth of DC to 2 Hz and fourth-order Sallen-Key cutoff, the design is ideally suited for various applications in the field of analytical chemistry and biology, such as laser-induced fluorescence detection or chemiluminescence measurements.

A handheld-type total integrated capillary electrophoresis system for SARS-CoV-2 diagnostics: Power, fluorescence detection, and data analysis by smartphone

A micro-capillary electrophoresis (μCE) system is one of the widely adopted techniques in the molecular diagnostics and DNA sequencing due to the benefits of high resolution, rapid analysis, and low reagent consumption, but due to the requirements of bulky high-power suppliers and an expensive laser-induced fluorescence detector module, the conventional set-up of μCE system is not adequate for point-of-care (POC) molecular diagnostics. In this study, we constructed a miniaturized and integrated μCE system which can be manipulated by a smartphone. The smartphone not only powers two boost converters and an excited laser, but also controls the relay for the power switch. Moreover, the complementary metal-oxide-semiconductor (CMOS) camera of the smartphone was used for detecting the fluorescence signal of amplicons amplified with reverse transcription-polymerase chain reaction (RT-PCR). We also developed a web-based application so that the raw data of the recorded fluorescence intensity versus the running time can display typical capillary electropherograms on the smartphone. The total size of the hand-held μCE system was 9.6 cm [Width] × 22 cm [Length] × 15.5 cm [Height], and the weight was ∼1 kg, which is suitable for POC DNA testing. In the integrated smartphone-associated μCE system, we could accurately analyze two genes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), namely N gene and S gene along with two bracket ladders in 6 min to identify SARS-CoV-2. Such an advanced μCE platform can be applied for a variety of on-site molecular diagnostics fields with user-friendliness.

Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans

BackgroundSulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans.ResultsOur screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO4 when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO4 (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated.ConclusionThe present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses.

Separation and determination of 3-hydroxyaspartate by online concentration capillary electrophoresis/laser-induced fluorescence with microwave-assisted derivatization.

A chiral analytical method was proposed based on capillary electrophoresis with laser-induced fluorescence detection coupled with microwave-assisted derivatization for the simultaneous baseline separation and sensitive detection of four stereoisomers of 3-hydroxyaspartate. The derivatization reaction of 3-hydroxyaspartate with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole was greatly accelerated by microwave irradiation. Under the optimized conditions, the derivatization yield was increased by 20% and the derivatization time was shortened by 20 min when compared with those from conventional water bath heating. In addition, the sensitivity was improved by online sample concentration methods. The detection limit of l-threo-3-hydroxyaspartate obtained by large-volume sample stacking with polarity switching was 5.3nmol/L, which was around 1000-fold lower than that of the capillary electrophoresis/laser-induced fluorescence without stacking. The excellent analytical performance in terms of linearity and precision was also achieved. Furthermore, the developed method was successfully applied to the determination of 3-hydroxyaspartate in the spiked urine, and satisfactory recoveries were obtained ranging from 90.5 to 107.0%.

Landé [formula omitted] factors of the electronic levels of the holmium atom

In the present work measurements of Landé gJ factors for some electronic levels of the holmium atom are presented. The measurements were performed with the use of the Zeeman effect of the hyperfine structure, observed with the method of laser spectroscopy in a hollow cathode discharge lamp placed in constant magnetic field, with laser induced fluorescence detection. Investigation of 69 spectral lines of holmium in the spectral range 490–670 nm enabled for the first time the determination of gJ factors for 15 levels belonging to the odd-parity configurations of Ho and 18 levels belonging to the even-parity configurations.

Watching a hydroperoxyalkyl radical (•QOOH) dissociate.

A prototypical hydroperoxyalkyl radical (•QOOH) intermediate, transiently formed in the oxidation of volatile organic compounds, was directly observed through its infrared fingerprint and energy-dependent unimolecular decay to hydroxyl radical and cyclic ether products. Direct time-domain measurements of •QOOH unimolecular dissociation rates over a wide range of energies were found to be in accord with those predicted theoretically using state-of-the-art electronic structure characterizations of the transition state barrier region. Unimolecular decay was enhanced by substantial heavy-atom tunneling involving O-O elongation and C-C-O angle contraction along the reaction pathway. Master equation modeling yielded a fully a priori prediction of the pressure-dependent thermal unimolecular dissociation rates for the •QOOH intermediate-again increased by heavy-atom tunneling-which are required for global models of atmospheric and combustion chemistry.

Spectral fingerprint of stabilized •QOOH

Chemical Kinetics Carbon-centered radicals containing the hydroperoxy group, commonly denoted as •QOOH, are elusive but are among the most critical intermediate species for kinetic modeling of hydrocarbon oxidation in various atmospheric and combustion processes. Their direct experimental observation is a long-standing challenge, with only one successful previous attempt. Using a combination of infrared activation spectroscopy and ultraviolet laser–induced fluorescence detection, Hansen et al. directly characterized the vibrational structure of a •QOOH intermediate in isobutane oxidation, collisionally stabilized and isolated, and followed its dissociative evolution under infrared activation with time and energy resolution. High-level electronic structure calculations revealed an important role of heavy-atom tunneling in this process. Science , abj0412, this issue p. [679][1] [1]: /lookup/doi/10.1126/science.abj0412

Capillary Electrophoresis-Indirect Laser-Induced Fluorescence Detection of Neomycin in Fish

Capillary Electrophoresis-Indirect Laser-Induced Fluorescence Detection of Neomycin in Fish

Multimaterial 3D-printed contactless conductivity/laser-induced fluorescence dual-detection cell for capillary electrophoresis

利用多材料3D打印技术研制了用于毛细管电泳(CE)的二合一检测池,实现了电容耦合非接触电导(C4D)与共聚焦激光诱导荧光(LIF)两种检测方法在毛细管柱上同一位置同时检测。3D打印的检测池采用了导电的复合聚乳酸(PLA)材料制作C4D的屏蔽层,采用普通的绝缘PLA材料支撑C4D金属管电极并隔离屏蔽层。两根金属管电极通过“打印-暂停-打印”的方式嵌入到检测池中,两电极被2 mm厚的导电屏蔽层隔开,在屏蔽层中有一直径为1 mm的圆形通孔用于LIF检测。该检测池与带流通式进样接口的自组装CE系统联用,用于同时检测无机离子和异硫氰酸荧光素(FITC)标记的氨基酸。研究优化了C4D激励信号频率与电泳缓冲液浓度,选用的电泳缓冲溶液为10 mmol/L 3-吗啉丙烷-1-磺酸(MOPS)与10 mmol/L二(2-羟乙基)亚氨基三(羟甲基)甲烷(Bis-Tris)的混合溶液,选用C4D激励频率为77 kHz。二合一检测池应用于内径为25 μm的毛细管时,C 4D对Na+、K+和Li+的检出限分别为2.2、2.0和2.6 μmol/L; LIF对荧光素和FITC的检出限分别为7.6和1.7 nmol/L。两种检测方法的相对标准偏差在0.3%至4.5%之间(n=3),工作曲线的相关系数r 2≥0.9904。采用3D打印技术可以在实验室内实现复杂结构的制作,降低了制作的成本,且便于方法的推广和改进。

Quantitative Capillary Electrophoresis for Analysis of Extracellular Vesicles (EVqCE)

Extracellular Vesicles (EVs) gained significant interest within the last decade as a new source of biomarkers for the early detection of diseases and a promising tool for therapeutic applications. In this work, we present Extracellular Vesicles Quantitative Capillary Electrophoresis (EVqCE) to measure an average mass of RNA in EVs, determine EV concentrations and the degree of EV degradation after sample handling. We used EVqCE to analyze EVs isolated from conditioned media of three cancer cell lines. EVqCE employs capillary zone electrophoresis with laser-induced fluorescent detection to separate intact EVs from free nucleic acids. After lysis of EVs with a detergent, the encapsulated nucleic acids are released. Therefore, the initial concentration of intact EVs is calculated based on a nucleic acid peak gain. EVqCE works in a dynamic range of EV concentrations from 108 to 1010 particles/mL. The quantification process can be completed in less than one hour and requires minimum optimization. Furthermore, the average mass of RNA was found to be in the range of 200–400 ag per particle, noting that more aggressive cancer cells have less RNA in EVs (200 ag per particle) than non-aggressive cancer cells (350 ag per particle). EVqCE works well for the degradation analysis of EVs. Sonication for 10 min at 40 kHz caused 85% degradation of EVs, 10 freeze-thaw cycles (from −80 °C to 22 °C) produced 40%, 14-day storage at 4 °C made 32%, and vortexing for 5 min caused 5% degradation. Presently, EVqCE cannot separate and distinguish individual EV populations (exosomes, microvesicles, apoptotic bodies) from each other. Still, it is tolerant to the presence of non-EV particles, protein-lipid complexes, and protein aggregates.

Integrated workflow for urinary prostate specific antigen N-glycosylation analysis using sdAb partitioning and downstream capillary electrophoresis separation

Prostate cancer represents the second highest malignancy rate in men in all cancer diagnoses worldwide. The development and progression of prostate cancer is not completely understood yet at molecular level, but it has been reported that changes in the N-glycosylation of prostate-specific antigen (PSA) occur during tumor genesis. In this paper we report on the development and implementation of a high-throughput capillary electrophoresis based glycan analysis workflow for urinary PSA analysis. The technology utilizes selective, high yield single domain antibody based PSA capture, followed by preconcentration and capillary electrophoresis coupled with laser-induced fluorescence detection, resulting in high resolution N-glycan profiles. Urinary PSA glycan profiles were compared to a commercially available PSA standard revealing differences in their α2,3- and α2,6-sialylated isomers, proving the excellent selectivity of the suggested workflow. This is important as sialylation classification plays an important role in the differentiation between indolent, significant and aggressive forms of prostate cancer.

Automated Capillary Electrophoresis System Compatible with Multiple Detectors for Potential In Situ Spaceflight Missions.

The in situ search for chemical signatures of life on extraterrestrial worlds requires automated hardware capable of performing detailed compositional analysis during robotic missions of exploration. The use of electrophoretic separations in this search is particularly powerful, enabling analysis of a wide range of soluble organic compounds potentially indicative of life, as well as inorganic compounds that can serve as indicators of habitability. However, to detect this broad range of compounds with a single electrophoresis instrument, a combination of different detection modes is required. For detection of any ionizable species, including organic compounds that do not participate in terrestrial biology (i.e., "unknown unknowns"), mass spectrometry (MS) is essential. Inorganic ions, or any dissolved charged species present, can be analyzed using capacitively coupled contactless conductivity detection (C4D). Additionally, for the trace analysis of compounds of key interest to astrobiology (particularly, amino acids), laser-induced fluorescence (LIF) detection holds unique promise, due to the fact that it has the highest demonstrated sensitivity of any form of detection. Here, we demonstrate a fully automated, portable capillary electrophoresis analyzer that is capable of all these modes of detection. The prototype system developed here addresses the three most significant challenges for doing electrophoretic separations: precise sample injection, HV isolation, and automation of all operational steps. These key challenges were successfully addressed with the use of custom-designed rotor-stator valves with optimized operational sequences incorporating gas purging steps, rinses, and HV application.