查看更多>>摘要：Nucleic acid detection technology is now widely used in scientific research and clinical testing, such as infectious and genetic diseases screening, molecular diagnosis of tumors and pharmacogenomic research, which is also an important part of in vitro diagnostics (IVD). However, with the increasing requirements of diagnosis and treatment, existing nucleic acid detection technologies are facing challenges in dealing with the current problems (especially since the outbreak of coronavirus disease in 2019 (Covid-19)). Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas)-based diagnostics have become a hot spot of attention. CRISPR/Cas has been developed as a molecular detection tool besides scientific research in biology and medicine fields, and some CRISPR-based products have already been translated. It is known as the "next-generation molecular diagnostic technology" because of its advantages such as easy design and accurate identification. CRISPR/Cas relies on pre-amplification of target sequences and subsequent detection of Cas proteins. Combining the CRISPR/Cas system with various isothermal nucleic acid amplification strategies can generate amplified detection signals, enrich low abundance molecular targets, improve the specificity and sensitivity of analysis, and develop point-of-care (POC) diagnostic techniques. In this review, we analyze the current status of CRISPR/Cas systems and isothermal amplification, report the advantages of combining the two and summarize the recent progress with the integration of both technologies with POC sensors in the nucleic acid field. In addition, the challenges and future prospects of CRISPR technology combined with isothermal amplification strategies in biosensing and clinical applications are discussed.
de Barros, Rachel MartinsLissalde, SophieGuibal, RobinGuibaud, Gilles...
8页
查看更多>>摘要：An analysis method for four families of hormones (estrogens, progestins, androgens and prostaglandins), dedicated to an efficient water monitoring with passive sampling, was developed using a liquid chromatography tandem mass spectrometry with triple quadrupole coupling and universal electrospray ionisation. Thirteen natural and synthetic hormones in ultra-pure water could be analysed in a single run according to the French Standard NF T90-210: calibration range of 0.1 (except for 17 beta-Estradiol, Estriol, Estrone and Diethylstilbestrol, from 0.5 mu g/L; and Ethinylestradiol, from 1 mu g/L) to 20 mu g/L with linear regressions (R2 >= 0.96), maximum accuracy deviations of 30% at intermediate fidelity for three concentration references (1, 10 and 20 mu g/L) and instrumental LOQs from 0.05 to 1 mu g/L. The stability of 11 hormones (10 mu g/L) was studied under several storage conditions and sample evaporation. All selected hormones were stable for 60 days at -18 degrees C, 7 days at 4 degrees C and 7 days at 20 degrees C but continued drying flow after evaporation should be avoided, especially for 17 alpha-Estradiol, Estrone and Diethylstilbestrol. Observed matrix effects using o-DGT extracts (diffusive gradient in thin-film sampler for polar organics) containing an environmental matrix varied from 24 to 92% but all matrix effects were corrected with IS use. Therefore, the developed method, coupled with o-DGT, was tested with the o-DGT deployment in rivers. Using diffusion coefficients from the literature or lab determined, the concentrations in the rivers varied for Estrone from 1.8 ng/L to 2.5 ng/L, and for Androstenedione from 0.4 to 1.1 ng/L.
查看更多>>摘要：Methionine oxidation is a reversible post-translational protein modification, affecting protein function, and implicated in aging and degenerative diseases. The detection of accumulating methionine oxidation in living cells or organisms, however, has not been achieved. Here we introduce a genetically encoded probe for methionine oxidation (GEPMO), based on the super-folder green fluorescent protein (sfGFP), as a specific, versatile, and integrating sensor for methionine oxidation. Placed at amino-acid position 147 in an otherwise methionine-less sfGFP, the oxidation of this specific methionine to methionine sulfoxide results in a ratiometric fluorescence change when excited with -400 and -470 nm light. The strength and homogeneity of the sensor expression is suited for live-cell imaging as well as fluorescence-activated cell sorting (FACS) experiments using standard laser wavelengths (405/488 nm). Expressed in mammalian cells and also in S. cerevisiae, the sensor protein faithfully reports on the status of methionine oxidation in an integrating manner. Variants targeted to membranes and the mitochondria provide subcellular resolution of methionine oxidation, e.g. reporting on site-specific oxidation by illumination of endogenous protoporphyrin IX.
查看更多>>摘要：Ambient mass spectrometry is used for direct analysis and high-throughput screening in many fields. However, most researches are about qualitative analysis. Quantitative detection based on AMS only performs on standard compounds and the relative standard deviation is so large that the accuracy of the result is low. In this study, a hydrogen flame ion source with ultrasonic nebulizer as sampling unit was established to enable solid samples to extract, nebulize and quantitatively detect in situ, with high sensitivity. This device was used to quantificationally determine the content of diisopropylnaphthalene (DIPN) in food packaging paper to identify recycled paper. Rapid analysis was performed in situ without complex pretreatment and the whole analysis time was less than 10 min. It's environmentally friendly that only 100 mg (or less) of sample and no more than 1 mL of solvent are required for one test. The external standard method was used for quantitative determination. The limit of detection was measured to be as low as 0.01 ng mL-1 and the linear dynamic range was 0.03-0.60 mu g mL-1 in positive multiple reactions monitoring mode. It has been successfully applied to detect actual samples and the content of DIPN was 0.020-0.095 mg kg-1.
查看更多>>摘要：Molecular identification is a fundamental issue in astrobiology to investigate the routes of emergence of life on our planet involving in particular a potential seeding of extraterrestrial organic matter on the primitive Earth. However, this project encompasses major difficulties due to the low concentration of molecules present in bodies of the Solar System. This work proposes an integrative analytical workflow, no longer based on GC-MS instruments, to enhance comprehensive analysis of organic markers in these objects. Our strategy combines UPLCHRMS and UPLC-MRM MS methods to bring both a broad molecular mapping and detailed data on indigenous compounds present in any extraterrestrial objects or laboratory analogs. Applied on water extracts from fresh meteorites, our workflow highlights a wide range of free molecules in the non-treated extracts and reveals the wide diversity of amino acid and nucleobase isomers that could lead to misinterpretation as far as the molecular composition of meteorite extracts cannot be anticipated. This strategy, never explored so far, would provide new clues for studying the organic matter in space and should offer new perspectives on its evolution and reactivity.
查看更多>>摘要：A novel molecularly imprinted monolithic (MIM) column was designed and fabricated using the epitope approach, and was used for the selective capillary microextraction (CME) of the neuropeptides neumtensin (NT) and neummedin N (NmN). The MIMs were synthesized in a capillary by thermally initiated polymerization of the functional monomer (methacrylic acid (MAA)), in the presence of a dummy template (Pro-Tyr-Ile-Leu (PYIL)), a crosslinker and porogens. The resulting monoliths were characterized by scanning electron microscopy, pore size distribution measurement, and Fourier transform infrared spectroscopy. Different synthesis conditions of the MIM column were investigated. The parameters affecting the MIM-CME performance, including loading, washing and elution protocols, were optimized as well. The MIMs were used to enrich NT and NmN from human plasma prior to HPLC-UV analysis. The imprinted monolith showed an excellent maximum adsorption capacity of 245-711 mg mL(-1) and selectivity (imprinting factor of 5.7-13.4) towards its target peptides. Low detection limits of 0.62 and 1.20 nM, and satisfactory recoveries (82.5-98.8%) were obtained for NT and NmN, respectively. The proposed MIM-CME/HPLC-UV method was found suitable to be used as an effective tool for the highly efficient and specific analysis of NT and NmN in human plasma samples.
查看更多>>摘要：Phenolic and substituted phenol based resoles are commonly used in the formulation of can coatings. However, migration analyses of these coatings are very little described compared to other coating technologies. While epoxy and polyester have well known migrants with defined formation mechanisms, Non-Intentionally Added Substances (NIAS) specifically related to the phenolic resin are hardly studied in the literature. The goal of the publication is to further explore the influence of the phenolic resole, used in the formulation of can coatings, on extracted NIAS's nature. Six different model polyester-phenolic can coatings were formulated each with a specific phenol, cresol or tertbutylphenol-based resole. Can coating films were extracted for 24 h at 40 degrees C in acetonitrile before analysis. NIAS identification was done using gas chromatography separation coupled to high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy analyses. Cyclic polyester oligomers were found in all extracts, with oligomers found in a range of 10 mu g/dm(2) to 226 mu g/dm(2), without specific influence of the resole used in formulation. While very few or no peaks were detected from cresol- and phenol-based resoles, 48 peaks were specifically observed in coating extracts of formulas with tertbutylphenol-based resoles as well as in their respective resoles. The most intense peaks were identified as aldehyde compounds by HRMS and NMR analysis. These aldehydes were semi-quantified in similar proportions as polyester oligomers. The presence of such aldehydes has never been reported in the literature regarding NIAS in can coatings. Further study will then be needed to better understand the aldehyde formation mechanism and assess the toxicological profile of such chemicals.
查看更多>>摘要：The recent development of hydrophobic deep eutectic solvents (HDESs) has led to growing interest in these reagents as possible environmentally benign replacements for conventional organic media in a host of applications, among them metal ion separations by liquid-liquid extraction. To evaluate the potential utility of these novel solvents in this application, a systematic examination of the facilitated transfer of selected alkali and alkaline earth cations into representative HDESs from aqueous solution in the presence of a macrocyclic polyether (i.e., a crown ether) has been undertaken. Comparison of the results to those obtained for a series of oxygenated, aliphatic solvents (n-alcohols) and for several 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) under the same conditions indicate that despite frequent suggestions that some HDESs resemble ILs, metal ion distribution in HDES-aqueous systems more closely mimics that seen for the alcohols. Metal ion partitioning in these systems appears less dependent on the water content of the organic phase and on structural variations in the solvent than is the case for either alcohols or ionic liquids, however. The implications of these results for the design and application of HDES-based extraction systems for metal ions are described.
查看更多>>摘要：Rapid and convenient preparation of covalent organic framework (COF) coated fibers is of great significance to solid-phase microextraction (SPME) technology. In this work, a novel chemical preparation strategy was established for rapid fabrication of beta-ketoenamine-linked COF coatings, in which clay-like starting materials of COFs were first wrapped on the fiber surface through self-viscosity and further fixed on the fiber in an oven via chemical bonding. Based on this strategy, four different COF (TpTph, TpPa-1, TpBD and TpTpb) coated fibers was fabricated within 1 h, which is very rapid compared to the recently reported research. Moreover, the strategy also demonstrates the good general applicability for COF fiber preparation. Subsequently, the TpTph coated fiber was used to develop a new SPME method for gas chromatography-tandem mass spectrometry (GC-MS/MS) of trace phthalic acid esters (PAEs) in environmental water. The developed analytical method compared to the previous SPME methods for PAEs based on other sorbents possesses low limits of detection (LODs, 0.02-0.08 ng L-1), and better or comparable precision (RSD <= 9.4%, n = 6), and relatively short extraction time. Furthermore, the trace PAEs (0.27-11.62 ng L-1) in the real water samples were successfully detected with recoveries of 82.2-117.5%. The above results indicates that the proposed fiber preparation strategy is reliable and opens a potential avenue for rapid and facile fabrication of COF coated fibers.
查看更多>>摘要：Carbon monoxide (CO), as one of significant gas transmitter, is closely associated with a variety of physiological and pathological processes. Although plenty of fluorescent probes have been prepared for detecting CO, most of them suffer from water-soluble fluorophores and short emission wavelength, which tends to diffuse and is limited to apply in vivo. Herein, a novel water-soluble fluorescent probe (HPQ-MQ-CO) is prepared to detect CO by releasing a precipitating fluorochrome (HPQ-MQ-OH), which is developed by introducing the 1-ethyl-2-methylquinoline group into HPQ to obtain long emission wavelength and good diffusion resistant ability. Allyl formate, as the identification unit of CO, has good water solubility and quenches the fluorescence of HPQ-MQ-CO. When the probe reacts with CO and Pd2+, an long-emission and solid-state fluorescence signal at 650 nm can be observed, which is based on excited-state intramolecular proton transfer (ESIPT) mechanism. When the concentration of CO is raised to 100.0 mu M, the fluorescence is increased 29 times, indicating the sensitivity of the probe. Moreover, this probe shows prominent selectivity for CO compared with other interfering species. Given these advantages, HPQ-MQ-CO can be used for CO detection in HepG2 cells and zebrafish by in-situ and longterm fluorescence imaging. In addition, this probe can monitor the up-regulation of CO in HepG2 cells and zebrafish during drug-induced liver injury (DILI).
NSTL
Elsevier