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Accumulated evidence has shown that Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with exposure to aristolochic acids (AA), and the associated DNA adduct formation, as a key causative factor of BEN development. Here, we show that coexposure to arsenic, cadmium, and iron increases the DNA adduct formation of AA in cultured kidney cells, while exhibiting both an exposure concentration and duration dependence. In contrast, coexposure to calcium and copper showed a decreasing DNA adduct formation. Because DNA damage is responsible for both the nephrotoxicity and carcinogenicity of AA, these results shed greater light on the endemic nature of BEN.
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Ácidos Aristolóquicos , Nefropatia dos Bálcãs , Metais Pesados , Humanos , Adutos de DNA , Ácidos Aristolóquicos/toxicidade , Nefropatia dos Bálcãs/induzido quimicamente , Metais Pesados/toxicidadeRESUMO
Tens of thousands of people in southern Europe suffer from Balkan endemic nephropathy (BEN), and four times as many are at risk. Incidental ingestion of aristolochic acids (AAs), stemming from the ubiquitousAristolochia clematitis(birthwort) weed in the region, leads to DNA adduct-induced toxicity in kidney cells, the primary cause of BEN. Numerous cofactors, including toxic organics and metals, have been investigated, but all have shown small contributions to the overall BEN relative to non-BEN village distribution gradients. Here, we reveal that combustion-derived pollutants from wood and coal burning in Serbia also contaminate arable soil and test as plausible causative factors of BEN. Using a GC-MS screening method, biomass-burning-derived furfural and coal-burning-derived medium-chain alkanes were detected in soil samples from BEN endemic areas levels at up to 63-times and 14-times higher, respectively, than in nonendemic areas. Significantly higher amounts were also detected in colocated wheat grains. Coexposure studies with cultured kidney cells showed that these pollutants enhance DNA adduct formation by AA, - the cause of AA nephrotoxicity and carcinogenicity. With the coincidence of birthwort-derived AAs and the widespread practice of biomass and coal burning for household cooking and heating purposes and agricultural burning in rural low-lying flood-affected areas in the Balkans, these results implicate combustion-derived pollutants in promoting the development of BEN.
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Nefropatia dos Bálcãs , Inundações , Nefropatia dos Bálcãs/induzido quimicamente , Nefropatia dos Bálcãs/epidemiologia , Humanos , Carvão Mineral , Sérvia , Poluentes do Solo/toxicidade , Ácidos Aristolóquicos , Animais , Aristolochia/química , Península Balcânica , Madeira , Nefropatias/induzido quimicamenteRESUMO
Microreactors have gained increasing attention in their application toward continuous micro flow synthesis. An unsolved problem of continuous flow synthesis is the lack of techniques for continuous product purification. Herein, we present a micro free-flow electrophoresis device and accompanying setup that enables the continuous separation and purification of unlabeled organic synthesis products. The system is applied to the separation and purification of triarylmethanes. For imaging of the unlabeled analytes on-chip a novel setup for large area (3.6 cm2) deep ultra violet excitation fluorescence detection was developed. Suitable separation conditions based on low conductivity electrophoresis buffers were devised to purify the product. With the optimized conditions, starting materials and product of the synthesis were well separated (R > 1.2). The separation was found to be very stable with relative standard deviations of the peak positions smaller than 3.5% over 15 min. The stable conditions enabled collection of the separated compounds, and purity of the product fraction was confirmed using capillary electrophoresis and mass spectrometry. This result demonstrates the great potential of free-flow electrophoresis as a technique for product purification or continuous clean-up in flow synthesis. Graphical Abstract Micro free-flow electrophoresis (µFFE) allows continuous separation and purification of small organic synthesis products. Enabled by a novel deep-UV imaging setup starting materials and product of a recently developed synthesis for triarylmethanes could be purified. Thereby demonstrating the potential of µFFE as continuous purification technique for micro flow synthesis.
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Optical absorbance detection based on attenuated light transmission is limited in sensitivity due to short path lengths in microfluidic and other miniaturized platforms. An alternative is detection using the photothermal effect. Herein we introduce a new kind of photothermal absorbance measurement using integrated luminescent temperature sensor spots inside microfluidic channels. The temperature sensors were photopolymerized inside the channels from NOA 81 UV-curable thiolene prepolymer doped with a tris(1,10-phenanthroline)ruthenium(II) temperature probe. The polymerized sensing structures were as small as 26 ± 3 µm in diameter and displayed a temperature resolution of better than 0.3 K between 20 and 50 °C. The absorbance from 532 nm laser excitation of the food dye Amaranth as a model analyte was quantified using these spots, and the influence of the flow rate, laser power, and concentration was investigated. Calibration yielded a linear relationship between analyte concentration and the temperature signal in the channels. The limit of detection for the azo-dye Amaranth (E123) in this setup was 13 µM. A minimal detectable absorbance of 3.2 × 10-3 AU was obtained using an optical path length of 125 µm in this initial study. A microreactor with integrated temperature sensors was then employed for an absorbance-based miniaturized nitrite analysis, yielding a detection limit of 26 µM at a total assay time of only 75 s. This technique is very promising for sensitive, and potentially spatially resolved, optical absorbance detection on the micro- and nanoscale.
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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.
Assuntos
Concentração de Íons de Hidrogênio , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Luminescência , Sondas Moleculares , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We demonstrate the fabrication, characterization and application of microfluidic chips capable of continuous electrophoretic separation via free flow isoelectric focussing (FFIEF). By integration of a near-infrared (NIR) fluorescent pH sensor layer under the whole separation bed, on-line observation of the pH gradient and determination of biomolecular isoelectric points (pI) was achieved within a few seconds. Using an optical setup for imaging of the intrinsic fluorescence of biomolecules at 266 nm excitation, labelling steps could be avoided and the native biomolecules could be separated, collected and analysed for their pI. The fabricated microchip was successfully used for the monitoring of the separation and simultaneous observation of the pH gradient during the isoelectric focussing of the proteins α-lactalbumin and ß-lactoglobulin, blood plasma proteins and the antibiotics ampicillin and ofloxacin. The obtained pIs are in good agreement with literature data, demonstrating the applicability of the system. Mass spectra from the separated antibiotics taken after 15 minutes of continuous separation from different fractions at the end of the microchip validated the separation via microfluidic isoelectric focussing and indicate the possibility of further on- or off-chip processing steps.
Assuntos
Ampicilina/isolamento & purificação , Antibacterianos/isolamento & purificação , Proteínas Sanguíneas/isolamento & purificação , Eletroforese em Microchip/instrumentação , Lactalbumina/isolamento & purificação , Lactoglobulinas/isolamento & purificação , Ofloxacino/isolamento & purificação , Animais , Desenho de Equipamento , Humanos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/instrumentação , Ponto IsoelétricoRESUMO
A strength of microfluidic chip laboratories is the rapid heat transfer that, in principle, enables a very homogeneous temperature distribution in chemical processes. In order to exploit this potential, we present an integrated chip system where the temperature is precisely controlled and monitored at the microfluidic channel level. This is realized by integration of a luminescent temperature sensor layer into the fluidic structure together with inkjet-printed micro heating elements. This allows steering of the temperature at the microchannel level and monitoring of the reaction progress simultaneously. A fabrication procedure is presented that allows for straightforward integration of thin polymer layers with optical sensing functionality in microchannels of glass-polydimethylsiloxane (PDMS) chips of only 150 µm width and 29 µm height. Sensor layers consisting of polyacrylonitrile and a temperature-sensitive ruthenium tris-phenanthroline probe with film thicknesses of about 0.5 to 6 µm were generated by combining blade coating and abrasion techniques. Optimal coating procedures were developed and evaluated. The chip-integrated sensor layers were calibrated and investigated with respect to stability, reproducibility, and response times. These microchips allowed observation of temperature in a wide range with a signal change of around 1.6 % per K and a maximum resolution of around 0.07 K. The device is employed to study temperature-controlled continuous micro flow reactions. This is demonstrated exemplarily for the tryptic cleavage of coumarin-modified peptides via fluorescence detection.
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Herein, the fabrication, characterization, calibration, and application of integrated microfluidic platforms for fast isoelectric point (pI) determinations via free-flow electrophoresis with integrated inkjet-printed fluorescent pH sensor microstructures are presented. These devices allow one to determine the pI of a biomolecule from a sample mixture with moderately good precision and without addition of markers in typically less than 10 s total separation and analysis time. Polyhydroxyethyl methacrylate (pHEMA) hydrogels were covalently coupled with fluorescein and hydroxypyrene trisulfonic acid (HPTS)-based pH probes. These were piezoelectrically jet-dispensed onto acrylate-modified glass as pH sensor microarrays with a diameter of 300-600 µm and thicknesses of 0.4-2.4 µm with high spatial accuracy. Microchip fabrication and integration of these pH sensor arrays was realized by multistep liquid-phase photolithography from oligoethylene glycol precursors resulting in glass-based microfluidic free-flow isoelectric focusing (µFFIEF) chips with integrated pH observation capabilities. The microchips were characterized with regard to pH sensitivity, response times, photo-, and flow stability. Depending on the sensor matrix, they allowed IEF within a pH range of roughly 5.5-10.5 with good sensitivity and fast response times. These microchips were used for FFIEF of small molecule markers and several protein mixtures with simultaneous monitoring of local pH. This allowed the determination of their pI via multispectral imaging of protein and pH sensor fluorescence without addition of external markers. Obtained pI's were generally in good agreement with known data, demonstrating the applicability of the method for pI determination in micropreparative procedures within a time frame of a few seconds only.
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The synthesis of Cs2AgIn0.9Bi0.1Cl6 nanocrystals was achieved at room temperature under ambient conditions using the ligand-assisted reprecipitation (LARP) method. The synthesized NCs exhibit bright orange emission when excited at 375 nm and have broad photoluminescence (PL) emission spectra with a maximum of 630 nm. A photoluminescence quantum yield (PLQY) of 36% was observed in these NCs without any polymer coatings. Polystyrene (PS), and poly (methyl methacrylate) (PMMA) were used to enhance the water stability and PLQY values up to 64%. Nanocomposite thin films with these polymer encapsulations exhibit good thermal stability up to at least 353 K and high quantum yields. PMMA-coated NCs showed long-term water stability for at least 4 months. The composites remain photostable when in contact with water for at least 120 min under continuous 365 nm UV illumination at 1 mW cm-2. Due to their excellent optical properties, aqueous stability, and wide range pH tolerance, these nanocomposite thin films could be employed for a variety of biological applications.
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In this study, we introduce time-resolved fluorescence detection with two-photon excitation at 532 nm for label-free analyte determination in microchip electrophoresis. In the developed method, information about analyte fluorescence lifetimes is collected by time-correlated single-photon counting, improving reliable peak assignment in electrophoretic separations. The determined limits of detection for serotonin, propranolol, and tryptophan were 51, 37, and 280 nM, respectively, using microfluidic chips made of fused silica. Applying two-photon excitation microchip separations and label-free detection could also be performed in borosilicate glass chips demonstrating the potential for label-free fluorescence detection in non-UV-transparent devices. Microchip electrophoresis with two-photon excited fluorescence detection was then applied for analyses of active compounds in plant extracts. Harmala alkaloids present in methanolic plant extracts from Peganum harmala could be separated within seconds and detected with on-the-fly determination of fluorescence lifetimes.
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We immobilized adherent human embryonic kidney (HEK) cells--which are able to trace adenosine triphosphate (ATP)--inside a microfluidic free-flow electrophoresis (µFFE) chip in order to develop an integrated device combining separation and biosensing capabilities. HEK 293 cells loaded with fluorescent calcium indicators were used as a model system to enable the spatially and temporally resolved detection of ATP. The local position of a 20 µM ATP stream was successfully visualized by these cells during free-flow electrophoresis, demonstrating the on-line detection capability of this technique towards native, unlabeled compounds.
Assuntos
Trifosfato de Adenosina/análise , Eletroforese/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Biossensoriais/instrumentação , Adesão Celular , Eletroforese/métodos , Desenho de Equipamento , Corantes Fluorescentes , Células HEK293 , Humanos , Técnicas Analíticas Microfluídicas/métodosRESUMO
Droplet microfluidic technology facilitates the development of high-throughput screening applications in nanoliter volumes. Surfactants provide stability for emulsified monodisperse droplets to carry out compartmentalization. Fluorinated silica-based nanoparticles are used; they can minimize crosstalk in microdroplets and provide further functionalities by surface labeling. Here we describe a protocol for monitoring pH changes in live single cells by fluorinated silica nanoparticles, for their synthesis, chip fabrication, and optical monitoring on the microscale. The nanoparticles are doped with ruthenium-tris-1,10-phenanthroline dichloride on the inside and conjugated with fluorescein isothiocyanate on the surface. This protocol may be used more generally to detect pH changes in microdroplets. The fluorinated silica nanoparticles can also be used as droplet stabilizers with an integrated luminescent sensor for other applications.
Assuntos
Ensaios de Triagem em Larga Escala , Dióxido de Silício , Reações Cruzadas , Fluoresceína , Concentração de Íons de HidrogênioRESUMO
In this work, we demonstrate an inexpensive method of prototyping microfluidics using a desktop injection molding machine. A centrifugal microfluidic device with a novel central filling mechanism was developed to demonstrate the technique. We overcame the limitations of desktop machines in replicating microfluidic features by variotherm heating and cooling the mold between 50 °C and 110 °C within two minutes. Variotherm heating enabled good replication of microfeatures, with a coefficient of variation averaging only 3.6% attained for the measured widths of 100 µm wide molded channels. Using this methodology, we produced functional polystyrene centrifugal microfluidic chips, capable of aliquoting fluids into 5.0 µL reaction chambers with 97.5% accuracy. We performed allele-specific loop-mediated isothermal amplification (AS-LAMP) reactions for genotyping CYP2C19 alleles on these chips. Readouts were generated using optical pH sensors integrated onto chips, by drop-casting sensor precursor solutions into reaction chambers before final chip assembly. Positive reactions could be discerned by decreases in pH sensor fluorescence, thresholded against negative control reactions lacking the primers for nucleic acid amplification and with time-to-results averaging 38 minutes. Variotherm desktop injection molding can enable researchers to prototype microfluidic devices more cost-effectively, in an iterative fashion, due to reduced costs of smaller, in-house molds. Designs prototyped this way can be directly translated to mass production, enhancing their commercialization potential and positive impacts.
Assuntos
Microfluídica , Técnicas de Amplificação de Ácido Nucleico , Técnicas de Amplificação de Ácido Nucleico/métodos , Dispositivos Lab-On-A-ChipRESUMO
High-throughput screening for optimal reaction conditions and the search for efficient catalysts is of eminent importance in the development of chemical processes and for expanding the spectrum of synthetic methodologies in chemistry. In this context we report a novel approach for a microfluidic chemical laboratory integrating organic synthesis, separation and time-resolved fluorescence detection on a single microchip. The feasibility of our integrated laboratory is demonstrated by monitoring the formation of tetrahydroisoquinoline derivatives by Pictet-Spengler condensation. After on-chip reaction the products and residual starting material were separated enantioselectively on the same chip. On-chip deep UV laser-induced fluorescence detection with time-correlated single photon counting was applied for compound assignment. The system was utilized to screen reaction conditions and various substrates for Pictet-Spengler reactions on-chip. Finally, the microlab was successfully applied to investigate enantioselective reactions using BINOL-based phosphoric acids as organocatalysts.
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Improved diagnostics are needed to manage the ongoing COVID-19 pandemic. In this study, we enhanced the color changes and sensitivity of colorimetric SARS-CoV-2 RT-LAMP assays based on triarylmethane dyes. We determined a mechanism for the color changes and obtained sensitivities of 10 RNA copies per microliter.
Assuntos
COVID-19 , SARS-CoV-2 , Colorimetria , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Pandemias , RNA Viral/genética , Reprodutibilidade dos TestesRESUMO
Microfluidic chips applied to the investigation of chirality allow reaction, separation and analysis of minuscule amounts of enantiomeric molecules. Chiral chip technology is employed in fields as diverse as pharmaceutical high throughput screening and deep space exploration missions.
RESUMO
Microchip electrophoresis with fluorescence detection has been applied for fast separation and determination of ephedra alkaloids in pharmaceutical formulations and body fluids. A custom epifluorescence microscope setup was employed and the compounds were separated within 40 s, allowing the detection of less than 200 ng/L for both analytes. Quantitation of the two stimulants was performed via a derivatization step using FITC without any extraction or preconcentration steps. The effects of different microchip types and excitation light sources were investigated and the method was successfully applied for the analysis of these compounds in tablet formulations, yielding recovery rates from 100.2 to 101.1% and relative standard deviations from 1.5 to 3.4%. Analysis of ephedrines was also carried out with human urine samples at detection limits of 500-1000 ng/L and relative standard deviations from 2.2 to 3.3% using argon ion LIF detection.
Assuntos
Alcaloides/análise , Alcaloides/química , Eletroforese em Microchip/métodos , Ephedra/química , Fluoresceína-5-Isotiocianato/análise , Pseudoefedrina/análise , Urina/química , Alcaloides/urina , Líquidos Corporais/química , Efedrina/análise , Efedrina/química , Efedrina/urina , Desenho de Equipamento , Fluorescência , Humanos , Técnicas Analíticas Microfluídicas/métodos , Preparações Farmacêuticas/análise , Pseudoefedrina/urina , Espectrometria de Fluorescência/métodos , Comprimidos/análiseRESUMO
Herein we introduce deep UV fluorescence lifetime detection in microfluidics applied for label-free detection and identification of various aromatic analytes in chip electrophoresis. For this purpose, a frequency quadrupled Nd:YAG (neodymium-doped yttrium aluminum garnet) picosecond laser at 266 nm was incorporated into an inverse fluorescence microscope setup with time-correlated single photon counting detection. This allowed recording of photon timing with sub-nanosecond precision. Thereby fluorescence decay curves are gathered on-the-fly and average lifetimes can be determined for each substance in the electropherogram. The aromatic compounds serotonin, propranolol, 3-phenoxy-1,2-propanediol and tryptophan were electrophoretically separated using a fused-silica microchip. Average lifetimes were independently determined for each compound via bi-exponential tail fitting. Time-correlated single photon counting also allows the discrimination of background fluorescence in the time domain. This results in improved signal-to-noise-ratios as demonstrated for the above model analytes. Microchip electrophoretic separations with fluorescence lifetime detection were also performed with a protein mixture containing lysozyme, trypsinogen and chymotrypsinogen emphasizing the potential for biopolymer analysis.
Assuntos
Eletroforese em Microchip/métodos , Espectrometria de Fluorescência/instrumentação , Glicerol/análogos & derivados , Glicerol/análise , Lasers de Estado Sólido , Éteres Fenílicos , Propranolol/análise , Reprodutibilidade dos Testes , Serotonina/análise , Razão Sinal-Ruído , Triptofano/análiseRESUMO
We present a fast and versatile method to produce functional micro free-flow electrophoresis chips. Microfluidic structures were generated between two glass slides applying multistep liquid-phase lithography, omitting troublesome bonding steps or cost-intensive master structures. Utilizing a novel spacer-less approach with the photodefinable polymer polyethyleneglycol dimethacrylate (PEG-DA), microfluidic devices with hydrophilic channels of only 25 µm in height were generated. The microfluidic chips feature ion-permeable segregation walls between the electrode channels and the separation bed and hydrophilic surfaces. The performance of the chip is demonstrated by free-flow electrophoretic separation of fluorescent xanthene dyes and fluorescently labeled amino acids.
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We present the first digital microfluidic (DMF) antimicrobial susceptibility test (AST) using an optical oxygen sensor film for in-situ and real-time continuous measurement of extracellular dissolved oxygen (DO). The device allows one to monitor bacterial growth across the entire cell culture area, and the fabricated device was utilized for a miniaturized and automated AST. The oxygen-sensitive probe platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin was embedded in a Hyflon AD 60 polymer and spin-coated as a 100 nm thick layer onto an ITO glass serving as the DMF ground electrode. This DMF-integrated oxygen sensing film was found to cause no negative effects to the droplet manipulation or cell growth on the chip. The developed DMF platform was used to monitor the DO consumption during Escherichia coli(E. coli) growth caused by cellular respiration. A rapid and reliable twofold dilution procedure was developed and performed, and the AST with E. coli ATCC 25922 in the presence of ampicillin, chloramphenicol, and tetracycline at different concentrations from 0.5 to 8 µg mL-1 was investigated. All sample dispensation, dilution, and mixing were performed automatically on the chip within 10 min. The minimum inhibitory concentration values measured from the DMF chip were consistent with those from the standard broth microdilution method but requiring only minimal sample handling and working with much smaller sample volumes.