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Paper-based devices are a portable, user-friendly, and affordable technology that is one of the best analytical tools for inexpensive diagnostic devices. Three-dimensional microfluidic paper-based analytical devices (3D-µPADs) are an evolution of single layer devices and they permit effective sample dispersion, individual layer treatment, and multiplex analytical assays. Here, we present the rational design of a wax-printed 3D-µPAD that enables more homogeneous permeation of fluids along the cellulose matrix than other existing designs in the literature. Moreover, we show the importance of the rational design of channels on these devices using glucose oxidase, peroxidase, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) reactions. We present an alternative method for layer stacking using a magnetic apparatus, which facilitates fluidic dispersion and improves the reproducibility of tests performed on 3D-µPADs. We also provide the optimized designs for printing, facilitating further studies using 3D-µPADs.
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The abundance of potentially habitable hypersaline environments in our solar system compels us to understand the impacts of high-salt matrices and brine dynamics on biosignature detection efforts. We identified and quantified organic compounds in brines from South Bay Salt Works (SBSW), where evapoconcentration of ocean water enables exploration of the impact of NaCl- and MgCl2-dominated brines on the detection of potential biosignature molecules. In SBSW, organic biosignature abundance and distribution are likely influenced by evapoconcentration, osmolyte accumulation, and preservation effects. Bioluminescence assays show that adenosine triphosphate (ATP) concentrations are higher in NaCl-rich, low water activity (aw) samples (<0.85) from SBSW. This is consistent with the accumulation and preservation of ATP at low aw as described in past laboratory studies. The water-soluble small organic molecule inventory was determined by using microchip capillary electrophoresis paired with high-resolution mass spectrometry (µCE-HRMS). We analyzed the relative distribution of proteinogenic amino acids with a recently developed quantitative method using CE-separation and laser-induced fluorescence (LIF) detection of amino acids in hypersaline brines. Salinity trends for dissolved free amino acids were consistent with amino acid residue abundance determined from the proteome of the microbial community predicted from metagenomic data. This highlights a tangible connection up and down the "-omics" ladder across changing geochemical conditions. The detection of water-soluble organic compounds, specifically proteinogenic amino acids at high abundance (>7 mM) in concentrated brines, demonstrates that potential organic biomarkers accumulate at hypersaline sites and suggests the possibility of long-term preservation. The detection of such molecules in high abundance when using diverse analytical tools appropriate for spacecraft suggests that life detection within hypersaline environments, such as evaporates on Mars and the surface or subsurface brines of ocean world Europa, is plausible and argues such environments should be a high priority for future exploration. Key Words: Salts-Analytical chemistry-Amino acids-Biosignatures-Capillary electrophoresis-Preservation. Astrobiology 24, 795-812.
Assuntos
Exobiologia , Meio Ambiente Extraterreno , Sais , Exobiologia/métodos , Meio Ambiente Extraterreno/química , Sais/análise , Sais/química , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Aminoácidos/análise , Salinidade , Compostos Orgânicos/análise , Voo Espacial , Água do Mar/química , Água do Mar/microbiologia , Água do Mar/análiseRESUMO
A fully integrated multilayer microfluidic chemical analyzer for automated sample processing and labeling, as well as analysis using capillary zone electrophoresis is developed and characterized. Using lifting gate microfluidic control valve technology, a microfluidic automaton consisting of a two-dimensional microvalve cellular array is fabricated with soft lithography in a format that enables facile integration with a microfluidic capillary electrophoresis device. The programmable sample processor performs precise mixing, metering, and routing operations that can be combined to achieve automation of complex and diverse assay protocols. Sample labeling protocols for amino acid, aldehyde/ketone and carboxylic acid analysis are performed automatically followed by automated transfer and analysis by the integrated microfluidic capillary electrophoresis chip. Equivalent performance to off-chip sample processing is demonstrated for each compound class; the automated analysis resulted in a limit of detection of ~16 nM for amino acids. Our microfluidic automaton provides a fully automated, portable microfluidic analysis system capable of autonomous analysis of diverse compound classes in challenging environments.
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Automação , Monitoramento Ambiental/instrumentação , Marte , Microfluídica/instrumentação , Eletroforese Capilar , Espectrometria de FluorescênciaRESUMO
We demonstrate microchip nonaqueous capillary electrophoresis (µNACE) analysis of primary aliphatic amines (C1-C18) in ethanol down to -20 °C as a first step in adapting microfluidic protocols for in situ analysis on Titan. To our knowledge, this is the first report of a nonaqueous separation at -20 °C on-chip. Limits of detection (LODs) ranged from 1.0 nM to 2.6 nM, and we identified several primary amines ranging in length from C2 to C16 in Titan aerosol analogue (tholin) samples; new amines were also detected in a tholin sample exposed to oxygen and liquid water. This preliminary work validates the sensitivity and efficacy of microfluidic chemical analysis of complex organics with relevance to Titan aerosols and surface deposits.
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Aminas/análise , Eletroforese em Microchip , Temperatura , SaturnoRESUMO
The detection of thiols on extraterrestrial bodies could provide evidence for life, as well as a host of potential prebiological or abiological processes. Here, we report a novel protocol to analyze organic thiols by microchip CE with LIF detection. Thiols were labeled with Pacific Blue C5 maleimide and analyzed by MEKC. The separation buffer consisted of 15 mM tetraborate pH 9.2 and 25 mM SDS. The optimized method provided LODs ranging from 1.4 to 15 nM. The method was validated using samples collected from geothermal pools at Hot Creek Gorge, California, which were found to contain 2-propanethiol and 1-butanethiol in the nanomolar concentration range. These samples serve as chemical analogues to material potentially present in the reducing environment of primitive Earth and also at sulfurous regions of Mars. Hence, the protocol developed here enables highly sensitive thiol analysis in samples with complexity comparable to that expected in astrobiologically relevant extraterrestrial settings. This new protocol could be readily added to the existing suite of microfluidic chemical analyses developed for in situ planetary exploration; all that is required is the incorporation of two new reagents to the payload of an existing instrument concept.
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Eletroforese em Microchip/métodos , Exobiologia/métodos , Compostos de Sulfidrila/análise , California , Fontes Termais/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Reprodutibilidade dos Testes , Dodecilsulfato de Sódio/química , Tensoativos/químicaRESUMO
The search for life in our solar system can be enabled by the characterization of extreme environments representing conditions expected on other planets within our solar system. Molecular abundances observed in these environments help establish instrument design requirements, including limits of detection and pH/salt tolerance, and may be used for validation of proposed planetary science instrumentation. Here, we optimize capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) separations for low limit of detection quantitative compositional analysis of amino acids in hypersaline samples using carboxyfluorescein succinimidyl ester (CFSE) as the amine-reactive fluorescent probe. Two methods were optimized for identification and quantification of proteinogenic amino acids, those with and those without acidic side chains, with limits of detection as low as 250 pM, improving on previous CFSE-amino acid CE-LIF methods by an order of magnitude. The resilience of the method to samples with high concentrations of Mg2+ (>4 M diluted to >0.4 M for analysis) is demonstrated on a sample collected from the salt harvesting facility South Bay Salt Works in San Diego, CA, demonstrating the highest Mg2+ tolerance for CE-LIF methods used in amino acid analyses to date. This advancement enables the rapid and robust analysis of trace amino acids and the search for biosignatures in hypersaline systems.
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The search for signs of life on extraterrestrial planetary bodies is among NASA's top priorities in Solar System exploration. The associated pursuit of organics and biomolecules as evidence of past or present life demands in situ investigations of planetary bodies for which sample return missions are neither practical nor affordable. These in situ studies require instrumentation capable of sensitive chemical analyses of complex mixtures including a broad range of organic molecules. Instrumentation must also be capable of autonomous operation aboard a robotically controlled vehicle that collects data and transmits it back to Earth. Microchip capillary electrophoresis (µCE) coupled to laser-induced fluorescence (LIF) detection provides this required sensitivity and targets a wide range of relevant organics while offering low mass, volume, and power requirements. Thus, this technology would be ideally suited for in situ studies of astrobiology targets, such as Mars, Europa, Enceladus, and Titan. In this review, we introduce the characteristics of these planetary bodies that make them compelling destinations for extraterrestrial astrobiological studies, and the principal groups of organics of interest associated with each. And although the technology we describe here was first developed specifically for proposed studies of Mars, by summarizing its evolution over the past decade, we demonstrate how µCE-LIF instrumentation has become an ideal candidate for missions of exploration to all of these nearby worlds in our Solar System.
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Eletroforese em Microchip/métodos , Exobiologia , Gravitação , Espectrometria de Fluorescência/métodosRESUMO
In situ analyses are essential to ascertain potential past or present habitability in celestial bodies. One technique that provides the sensitivity and miniaturization needed to successfully detect trace organics in the outer Solar System is laser-induced fluorescence (LIF) detection, which, when coupled with microfluidic systems, provides a powerful wet chemistry platform that can meet the size and resource consumption constraints of a remote analysis mission. Herein, a portable LIF detection module (44-mm long, 18-mm wide) was prototyped and utilized to quantify bulk organics in a liquid sample via manual and automated analysis utilizing a programmable microfluidic architecture. The experimental limit of detection (LOD) for primary amines was 11.8 µM. A sample (Y31B) collected from the Atacama Desert in Yungay, Chile, was analyzed manually and found to contain 300 ± 50 µM of bulk primary amine organics, while the automated microfluidic protocol found the sample to contain 289 ± 4 µM of primary amines. Automated analyses showed no statistically significant differences when compared to the manual analyses (t-test, C.I. 95%). Our results demonstrate that the coupling of programmable microfluidic devices with a custom lens tube-based LIF detector enables automated analysis of primary amines using a protocol appropriate for remote analyses. This technique is an invaluable tool for in situ analysis applications in distant, resource-restricted environments.
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Aminas , Técnicas Analíticas Microfluídicas , Aminas/análise , Dispositivos Lab-On-A-Chip , Lasers , Microfluídica/métodosRESUMO
Despite multiple orbiter and landed missions to extraterrestrial bodies in the solar system, including Mars and Titan, we still know relatively little about the detailed chemical composition and quantity of organics and biomolecules in those bodies. For chemical analysis on astrobiologically relevant targets such as Mars, Europa, Titan, and Enceladus, instrumentation should be extremely sensitive and capable of analyzing a broad range of organic molecules. Microchip capillary electrophoresis (µCE) with laser-induced fluorescence (LIF) detection provides this required sensitivity and targets a wide range of relevant markers but, to date, has lacked the necessary degree of automation for spaceflight applications. Here we describe a fully integrated microfluidic device capable of performing automated end-to-end analyses of amino acids by µCE with LIF detection. The device integrates an array of pneumatically actuated valves and pumps for autonomous fluidic routing with an electrophoretic channel. Operation of the device, including manipulation of liquids for sample pretreatment and electrophoretic analysis, was performed exclusively via computer control. The device was validated by mixing of laboratory standards and labeling of amino acids with Pacific Blue succinimidyl ester followed by electrophoretic analysis. To our knowledge, this is the first demonstration of completely automated end-to-end µCE analyses on a single, fully integrated microfluidic device.
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Aminoácidos/análise , Automação , Técnicas Analíticas MicrofluídicasRESUMO
The Multichannel Mars Organic Analyzer (McMOA), a portable instrument for the sensitive microchip capillary electrophoresis (CE) analysis of organic compounds such as amino acid biomarkers and polycyclic aromatic hydrocarbons (PAHs), is developed. The instrument uses a four-layer microchip, containing eight CE analysis systems integrated with a microfluidic network for autonomous fluidic processing. The McMOA has improved optical components that integrate 405 nm laser excitation with a linear-scanning optical system capable of multichannel real-time fluorescence spectroscopic analysis. The instrumental limit of detection is 6 pM (glycine). Microfluidic programs are executed to perform the automated sequential analysis of an amine-containing sample in each channel as well as eight consecutive analyses of alternating samples on the same channel, demonstrating less than 1% cross-contamination. The McMOA is used to identify the unique fluorescence spectra of nine components in a PAH standard and then applied to the analysis of a sediment sample from Lake Erie. The presence of benzo[a]pyrene and perylene in this sample is confirmed, and a peak coeluting with anthanthrene is disqualified based on spectral analysis. The McMOA exploits lab-on-a-chip technologies to fully integrate complex autonomous operations demonstrating the facile engineering of microchip-CE platforms for the analysis of a wide variety of organic compounds in planetary exploration.
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Aminoácidos/isolamento & purificação , Eletroforese em Microchip/instrumentação , Hidrocarbonetos Policíclicos Aromáticos/isolamento & purificação , Biomarcadores/análise , Desenho de Equipamento , Limite de DetecçãoRESUMO
A microchip CE method is developed for the analysis of two oxidized forms of carbon, aldehydes and ketones, with the Mars Organic Analyzer (MOA). Fluorescent derivitization is achieved in â¼ 15 min by hydrazone formation with Cascade Blue hydrazide in 30 mM borate pH 5-6. The microchip CE separation and analysis method is optimized via separation in 30 mM borate buffer, pH 9.5, at 20°C. A carbonyl standard consisting of ten aldehydes and ketones found in extraterrestrial matter is successfully separated; the resulting LOD depends on the reactivity of the compound and range from 70 pM for formaldehyde to 2 µM for benzophenone. To explore the utility of this method for analyzing complex samples, analyses of several fermented beverages are conducted, identifying ten aldehydes and ketones ranging from 30 nM to 5 mM. A Martian regolith simulant sample, consisting of a basalt matrix spiked with soluble ions and acetone, is designed and analyzed, but acetone is found to have a limited detectable lifetime under simulant Martian conditions. This work establishes the capability of the MOA for studying aldehydes and ketones, a critical class of oxidized organic molecules of interest in planetary and in terrestrial environmental and health studies.
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Aldeídos/análise , Biomarcadores/análise , Eletroforese em Microchip/métodos , Cetonas/análise , Modelos Químicos , Boratos/química , Exobiologia/métodos , Concentração de Íons de Hidrogênio , Marte , Compostos Organometálicos/química , Compostos Organofosforados/química , Solo/químicaRESUMO
Micro total analytical systems (µTAS) are attractive to multiple fields that include chemistry, medicine and engineering due to their portability, low power usage, potential for automation, and low sample and reagent consumption, which in turn results in low waste generation. The development of fully-functional µTAS is an iterative process, based on the design, fabrication and testing of multiple prototype microdevices. Typically, microfabrication protocols require a week or more of highly-skilled personnel time in high-maintenance cleanroom facilities, which makes this iterative process cost-prohibitive in many locations worldwide. Rapid-prototyping tools, in conjunction with the use of polydimethylsiloxane (PDMS), enable rapid development of microfluidic structures at lower costs, circumventing these issues in conventional microfabrication techniques. Multiple rapid-prototyping methods to fabricate PDMS-based microfluidic devices have been demonstrated in literature since the advent of soft-lithography in 1998; each method has its unique advantages and drawbacks. Here, we present a tutorial discussing current rapid-prototyping techniques to fabricate PDMS-based microdevices, including soft-lithography, print-and-peel and scaffolding techniques, among other methods, specifically comparing resolution of the features, fabrication processes and associated costs for each technique. We also present thoughts and insights towards each step of the iterative microfabrication process, from design to testing, to improve the development of fully-functional PDMS-based microfluidic devices at faster rates and lower costs.
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Microcapillary electrophoresis (µCE) enables high-resolution separations in miniaturized, automated microfluidic devices. Pairing this powerful separation technique with laser-induced fluorescence (LIF) enables a highly sensitive, quantitative, and compositional analysis of organic molecule monomers and short polymers, which are essential, ubiquitous components of life on Earth. Improving methods for their detection has applications to multiple scientific fields, particularly those related to medicine, industry, and space science. Here, a modular benchtop system using µCE with LIF detection was constructed and tested by analyzing standard amino acid samples of valine, serine, alanine, glycine, glutamic acid, and aspartic acid in multiple borate buffered solutions of increasing concentrations from 10 mM to 50 mM, all pH 9.5. The 35 mM borate buffer solution generated the highest resolution before Joule heating dominated. The limits of detection of alanine and glycine using 35 mM borate buffer were found to be 2.12 nM and 2.91 nM, respectively, comparable to other state-of-the-art µCE-LIF instruments. This benchtop system is amenable to a variety of detectors, including a photomultiplier tube, a silicon photomultiplier, or a spectrometer, and currently employs a spectrometer for facile multi-wavelength detection. Furthermore, the microdevice is easily exchanged to fit the desired application of the system, and optical components within the central filter cube can be easily replaced to target alternative fluorescent dyes. This work represents a significant step forward for the analysis of small organic molecules and biopolymers using µCE-LIF systems.
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The Mars Organic Analyzer (MOA), a portable microchip capillary electrophoresis (CE) instrument developed for sensitive amino acid analysis on Mars, is used to analyze laboratory standards and real-world samples for polycyclic aromatic hydrocarbons (PAHs). The microfabricated CE separation and analysis method for these hydrophobic analytes is optimized, resulting in a separation buffer consisting of 10 mM sulfobutylether-beta-cyclodextrin, 40 mM methyl-beta-cyclodextrin, 5 mM carbonate buffer at pH 10, 5 degrees C. A PAH standard consisting of seven PAHs found in extraterrestrial matter and two terrestrial PAHs is successfully baseline separated. Limits of detection for the components of the standard ranged from 2000 ppm to 6 ppb. Analysis of an environmental contamination standard from Lake Erie and of a hydrothermal vent chimney sample from the Guaymas Basin agreed with published composition. A Martian analogue sample from the Yungay Hills region of the Atacama Desert was analyzed and found to contain 9,10-diphenylanthracene, anthracene, anthanthrene, fluoranthene, perylene, and benzo[ghi]fluoranthene at ppm levels. This work establishes the viability of the MOA for detecting and analyzing PAHs in in situ planetary exploration.
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The fluorescent amine reactive probe Pacific Blue succinimidyl ester (PB) is used for the detection of trace amounts of amines and amino acids by microchip capillary electrophoresis on the Mars Organic Analyzer (MOA). The spectral and chemical properties of PB provide a 200-fold increase in sensitivity and improved resolution compared to fluorescamine derivatization. With the use of cross injection and PB labeling, the MOA detected amino acids at concentrations as low as 75 pM (sub-parts-per-trillion). Micellar electrokinetic chromatography (MEKC) which separates PB-labeled amino acids by their hydrophobicity is also demonstrated. The optimized MEKC conditions (45 mM CHAPSO, pH 6 at 5 degrees C) effectively separated amines and 25 amino acids with enantiomeric resolution of alanine, serine, and citrulline. Samples from the Yungay Hills region in the Atacama Desert, Chile, and from the Murchison meteorite are successfully analyzed using both techniques, and amino acids are found in the parts-per-billion range. Abiotic amino acids such as beta-alanine and epsilon-aminocaprioc acid are detected along with several neutral and acidic amino acids in the Murchison sample. The Atacama Desert sample is found to contain homochiral L-alanine and L-serine indicating the presence of extant or recently extinct life.
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Aminas/análise , Aminoácidos/análise , Eletroforese em Microchip/métodos , Corantes Fluorescentes/química , Marte , Succinimidas/química , Cromatografia , Clima Desértico , Exobiologia , Cinética , Meteoroides , Micelas , Sensibilidade e EspecificidadeRESUMO
Paper-based devices are a leading alternative among the main analytical tools for point-of-care testing, due to their portability, low-cost, and ease-of-use. Colorimetric readouts are the most common method of detection in these microfluidic devices, enabling qualitative, semi-quantitative and fully quantitative analysis of multiple analytes. There is a multitude of ways to obtain a colorimetric output in such devices, including nanoparticles, dyes, redox and pH indicators, and each has unique drawbacks and benefits. There are also multiple variables that impact the analysis of colorimetric reactions in microfluidic paper-based systems, including color homogeneity, image capture methods, and the data handling itself. Here, we present a critical review of recent developments and challenges of colorimetric detection on microfluidic paper-based analytical devices (µPADs), and present thoughts and insights towards future perspectives in the area to improve the use of colorimetric readouts in conjunction with µPADs.
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Colorimetria , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Papel , Bioensaio , Técnicas e Procedimentos Diagnósticos , Microfluídica , NanopartículasRESUMO
Enceladus presents an excellent opportunity to detect organic molecules that are relevant for habitability as well as bioorganic molecules that provide evidence for extraterrestrial life because Enceladus' plume is composed of material from the subsurface ocean that has a high habitability potential and significant organic content. A primary challenge is to send instruments to Enceladus that can efficiently sample organic molecules in the plume and analyze for the most relevant molecules with the necessary detection limits. To this end, we present the scientific feasibility and engineering design of the Enceladus Organic Analyzer (EOA) that uses a microfluidic capillary electrophoresis system to provide sensitive detection of a wide range of relevant organic molecules, including amines, amino acids, and carboxylic acids, with ppm plume-detection limits (100 pM limits of detection). Importantly, the design of a capture plate that effectively gathers plume ice particles at encounter velocities from 200 m/s to 5 km/s is described, and the ice particle impact is modeled to demonstrate that material will be efficiently captured without organic decomposition. While the EOA can also operate on a landed mission, the relative technical ease of a fly-by mission to Enceladus, the possibility to nondestructively capture pristine samples from deep within the Enceladus ocean, plus the high sensitivity of the EOA instrument for molecules of bioorganic relevance for life detection argue for the inclusion of EOA on Enceladus missions. Key Words: Lab-on-a-chip-Organic biomarkers-Life detection-Planetary exploration. Astrobiology 17, 902-912.
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Programmable microfluidic platforms (PMPs) are enabling significant advances in the utility of microfluidics for chemical and biochemical analysis. Traditional microfluidic devices are analogous to application-specific devices--a new device is needed to implement each new chemical or biochemical assay. PMPs are analogous to digital electronic processors--all that is needed to implement a new assay is a change in the order of operations conducted by the device. In this review, we introduce PMPs based on normally-closed microvalves. We discuss recent applications of PMPs in diverse fields including genetic analysis, antibody-based biomarker analysis, and chemical analysis in planetary exploration. Prospects, challenges, and future concepts for this emerging technology will also be presented.
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Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , HumanosRESUMO
Microchip capillary electrophoresis with laser-induced fluorescence detection (µCE-LIF) enables sensitive analyses of a wide range of analytes employing small volumes of sample and reagent (nL to µL) on an instrument platform with minimal mass, volume, and power requirements. This technique has been used previously in the analysis of amino acids and other organic molecules of interest in the fields of astrobiology and planetary science. Here, we present a protocol for the analysis of thiols using µCE-LIF. This protocol utilizes Pacific Blue C5-maleimide for fluorescent derivatization of thiols, enabling limits of detection in the low nM range (1.4-15 nM). Separations are conducted in micellar electrokinetic chromatography mode with 25 mM sodium dodecyl sulfate in 15 mM tetraborate, pH 9.2. This method allows analysis of 12 thiols in less than 2 min following a labeling step of 2 h. A step-by-step protocol, including tips on microchip capillary electrophoresis, is described here.
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Eletroforese Capilar/métodos , Eletroforese em Microchip/métodos , Compostos de Sulfidrila/análiseRESUMO
Fluorescent probes enable detection of otherwise nonfluorescent species via highly sensitive laser-induced fluorescence. Organic amines are predominantly nonfluorescent and are of analytical interest in agricultural and food science, biomedical applications, and biowarfare detection. Alexa Fluor 488 N-hydroxysuccinimidyl ester (AF488 NHS-ester) is an amine-specific fluorescent probe. Here, we demonstrate low limit of detection of long-chain (C9 to C18) primary amines and optimize AF488 derivatization of long-chain primary amines. The reaction was found to be equally efficient in all solvents studied (dimethylsulfoxide, ethanol, and N,N-dimethylformamide). While an organic base (N,N-diisopropylethylamine) is required to achieve efficient reaction between AF488 NHS-ester and organic amines with longer hydrophobic chains, high concentrations (>5 mM) result in increased levels of ethylamine and propylamine in the blank. Optimal incubation times were found to be >12 hrs at room temperature. We present an initial capillary electrophoresis separation for analysis using a simple micellar electrokinetic chromatography (MEKC) buffer consisting of 12 mM sodium dodecylsulfate (SDS) and 5 mM carbonate, pH 10. Limits of detection using the optimized labeling conditions and these separation conditions were 5-17 nM. The method presented here represents a novel addition to the arsenal of fluorescent probes available for highly sensitive analysis of small organic molecules.