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BACKGROUND: The early allopolyploid Brassica napus was a hybrid of two Brassica species, that had undergone a whole genome duplication event followed by genome restructuring, including deletions and small scale duplications. A large number of homologous genes appeared functional divergence during species domestication. Due to the high conservation of de novo glycerolipid biosynthesis, multiple homologues of glycerol-3-phosphate acyltransferases (GPATs) have been found in B. napus. Moreover, the functional variances among these homologous GPAT-encoding genes are unclear. RESULTS: In this study, four B. napus homologous genes encoding glycerol-3-phosphate acyltransferase 9 (BnaGPAT9) were characterized. Although a bioinformatics analysis indicated high protein sequence similarity, the homologues demonstrated tissue-specific expression patterns and functional divergence. Yeast genetic complementation assays revealed that BnaGPAT9-A1/C1 homologues but not BnaGPAT9-A10/C9 homologues encoded functional GPAT enzymes. Furthermore, a single nucleotide polymorphism of BnaGPAT9-C1 that occurred during the domestication process was associated with enzyme activity and contributed to the fatty acid composition. The seed-specific expression of BnGPAT9-C11124A increased the erucic acid content in the transformant seeds. CONCLUSIONS: This study revealed that BnaGPAT9 gene homologues evolved into functionally divergent forms with important roles in erucic acid biosynthesis.
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Brassica napus , Ácidos Erucicos , Glicerol , Glicerol-3-Fosfato O-Aciltransferasa , Saccharomyces cerevisiae , Semillas , FosfatosRESUMEN
Nanopores have developed into powerful single-molecule sensors capable of identifying and characterizing small polymers, such as DNA, by electrophoretically driving them through a nanoscale pore and monitoring temporary blockades in the ionic pore current. However, the relationship between nanopore signals and the physical properties of DNA remains only partly understood. Herein, we introduce a programmable DNA carrier platform to capture carefully designed DNA nanostructures. Controlled translocation experiments through our glass nanopores allowed us to disentangle this relationship. We vary DNA topology by changing the length, strand duplications, sequence, unpaired nucleotides, and rigidity of the analyte DNA and find that the ionic current drop is mainly determined by the volume and flexibility of the DNA nanostructure in the nanopore. Finally, we use our understanding of the role of DNA topology to discriminate circular single-stranded DNA molecules from linear ones with the same number of nucleotides using the nanopore signal.
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Nanoporos , ADN/química , Nucleótidos/química , Nucleótidos/genética , Nanotecnología , ADN de Cadena SimpleRESUMEN
Nanopore analysis relies on ensemble averaging of translocation signals obtained from numerous molecules, requiring a relatively high sample concentration and a long turnaround time from the sample to results. The recapture and subsequent re-reading of the same molecule is a promising alternative that enriches the signal information from a single molecule. Here, we describe how an asymmetric nanopore improves molecular ping-pong by promoting the recapture of the molecule in the trans reservoir. We also demonstrate that the molecular recapture could be improved by linking the target molecule to a long DNA carrier to reduce the diffusion, thereby achieving over 100 recapture events. Using this ping-pong methodology, we demonstrate its use in accurately resolving nanostructure motifs along a DNA scaffold through repeated detection. Our method offers novel insights into the control of DNA polymer dynamics within nanopore confinement and opens avenues for the development of a high-fidelity DNA detection platform.
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Nanoporos , ADN/química , Nanotecnología , Difusión , PolímerosRESUMEN
Multiplexed nucleic acid sensing methods with high specificity are vital for clinical diagnostics and infectious disease control, especially in the postpandemic era. Nanopore sensing techniques have developed in the past two decades, offering versatile tools for biosensing while enabling highly sensitive analyte measurements at the single-molecule level. Here, we establish a nanopore sensor based on DNA dumbbell nanoswitches for multiplexed nucleic acid detection and bacterial identification. The DNA nanotechnology-based sensor switches from an "open" into a "closed" state when a target strand hybridizes to two sequence-specific sensing overhangs. The loop in the DNA pulls two groups of dumbbells together. The change in topology results in an easily recognized peak in the current trace. Simultaneous detection of four different sequences was achieved by assembling four DNA dumbbell nanoswitches on one carrier. The high specificity of the dumbbell nanoswitch was verified by distinguishing single base variants in DNA and RNA targets using four barcoded carriers in multiplexed measurements. By combining multiple dumbbell nanoswitches with barcoded DNA carriers, we identified different bacterial species even with high sequence similarity by detecting strain specific 16S ribosomal RNA (rRNA) fragments.
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Técnicas Biosensibles , Nanoporos , Ácidos Nucleicos , ADN , Nanotecnología/métodos , Técnicas Biosensibles/métodosRESUMEN
While the solid-state nanopore shows increasing potential during sensitive and label-free single molecular analysis, target concentration and signal amplification method is in urgent need. In this article, a solution via designing a model nucleic acid circuit reaction that can produce "Y" shape-structure three-way DNA oligomers with controllable size and polymerization degree is proposed. Such a so-called lego-like three-way catalytic hairpin assembly (LK-3W-CHA) can provide both concentration amplification (via CHA circuit) and programmable size control (via lego-like building mode) to enhance spatiotemporal resolution in single molecular sensing of solid-state nanopore. Oligomers containing 1-4 DNA three-way junctions (Y monomers, Y1-Y4) are designed in proof-of-concept experiments and applications. When the oligomers are applied to direct translocation measurements, Y2-Y4 can significantly increase the signal resolution and stability than that of Y1. Meanwhile, Y1 to Y4 can be used as the tags on the long DNA carrier to provide very legible secondary signals for specific identification, multiple assays, and information storage. Compared with other possible tags, Y1-Y4 provides higher signal density and amplitude, and quasi-linear "inner reference" for each other, which may provide more systematic, reliable, and controllable experimental results.
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Técnicas Biosensibles , ADN , ADN/química , Transducción de Señal , Replicación del ADN , Técnicas Biosensibles/métodos , Límite de DetecciónRESUMEN
Assembly of DNA structures based on hybridization like split G-quadruplex (GQ) have great potential for the base-pair specific identification of nucleic acid targets. Herein, we combine multiple split G-quadruplex (GQ) assemblies on designed DNA nanostructures (carrier) with a solid-state nanopore sensing platform. The split GQ probes recognize various nucleic acid sequences in a parallel assay that is based on glass nanopore analysis of molecular structures. Specifically, we split a GQ into two asymmetric parts extended with sequences complementary to the target. The longer G-segment is in solution, and the shorter one is on a DNA carrier. If the target is present, the two separate GQ parts will be brought together to facilitate the split GQ formation and enhance the nanopore signal. We demonstrated detection of multiple target sequences from different viruses with low crosstalk. Given the programmability of this DNA based nanopore sensing platform, it is promising in biosensing.
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Técnicas Biosensibles , G-Cuádruplex , Nanoporos , Ácidos Nucleicos , ADN/química , Hibridación de Ácido NucleicoRESUMEN
BACKGROUND: Genetic improvement of soybean oil content depends on in-depth study of the glycerolipid biosynthesis pathway. The first acylation reaction catalysed by glycerol-3-phosphate acyltransferase (GPAT) is the rate-limiting step of triacylglycerol biosynthesis. However, the genes encoding GPATs in soybean remain unknown. METHODS: We used a novel yeast genetic complementation system and seed-specific heterologous expression to identify GmGPAT activity and molecular function in glycerolipid biosynthesis. RESULTS: Sixteen GmGPAT genes were cloned by reverse transcription-PCR for screening in yeast genetic complementation. The results showed that GmGPAT9-2 could restore the conditional lethal double knockout mutant strain ZAFU1, and GmGPAT1-1 exhibited low acyltransferase activity in serial dilution assays. In addition, the spatiotemporal expression pattern of GmGPAT9-2 exhibited tissue specificity in leaves, flowers and seeds at different developmental stages. Furthermore, both the proportion of arachidic acid and erucic acid were significantly elevated in Arabidopsis thaliana transgenic lines containing the seed-specific GmGPAT9-2 compared wild type, but the oil content was not affected. CONCLUSION: Together, our results provide reference data for future engineering of triacylglycerol biosynthesis and fatty acid composition improvement through GPATs in soybean.
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Arabidopsis , Glycine max , Aciltransferasas/genética , Aciltransferasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Ácidos Grasos/metabolismo , Glicerol/metabolismo , Fosfatos , Aceites de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Semillas/metabolismo , Aceite de Soja/análisis , Aceite de Soja/metabolismo , Glycine max/genética , Glycine max/metabolismo , Triglicéridos/metabolismoRESUMEN
The toehold-mediated strand displacement reaction (SDR) is a powerful enzyme-free tool for molecular manipulation, DNA computing, signal amplification, etc. However, precise modulation of SDR kinetics without changing the original design remains a significant challenge. We introduce a new means of modulating SDR kinetics using an external stimulus: a water-soluble FeII4L4 tetrahedral cage. Our results show that the presence of a flexible phosphate group and a minimum toehold segment length are essential for FeII4L4 binding to DNA. SDRs mediated by toehold ends in different lengths (3-5) were investigated as a function of cage concentration. Their reaction rates all first increased and then decreased as cage concentration increased. We infer that cage binding on the toehold end slows SDR, whereas the stabilization of intermediates that contain two overhangs accelerates SDR. The tetrahedral cage thus serves as a versatile tool for modulation of SDR kinetics.
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Deoxyribonucleic acid (DNA) nanostructure-based data encoding is an emerging information storage mode, offering rewritable, editable, and secure data storage. Herein, a DNA nanostructure-based storage method established on a solid-state nanopore sensing platform to save and encrypt a 2D grayscale image is proposed. DNA multi-way junctions of different sizes are attached to a double strand of DNA carriers, resulting in distinct levels of current blockades when passing through a glass nanopore with diameters around 14 nm. The resulting quaternary encoding doubles the capacity relative to a classical binary system. Through toehold-mediated strand displacement reactions, the DNA nanostructures can be precisely added to and removed from the DNA carrier. By encoding the image into 16 DNA carriers using the quaternary barcodes and reading them in one simultaneous measurement, the image is successfully saved, encrypted, and recovered. Avoiding any proteins or enzymatic reactions, the authors thus realize a pure DNA storage system on a nanopore platform with increased capacity and programmability.
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Nanoporos , ADN , Código de Barras del ADN Taxonómico , VidrioRESUMEN
Three-dimensional mesoporous lepidocrocite-type layered sodium titanate (LST) was constructed at room temperature by the electrostatic interaction between Ti1-δO24δ- nanosheets and Na+ ions. The results of a systematic X-ray diffraction investigation manifested the transition from the Ti1-δO24δ- nanosheets phase to the titanate/titania phase, which determined a phase diagram as a function of the temperature and NaCl concentration. In addition, scanning electron microscopy, inductively coupled plasma-mass spectrometry, thermogravimetric and differential thermal, N2 adsorption-desorption, Raman spectroscopy, Fourier transform infrared spectroscopy, as well as ζ-potential analyses were utilized for adequate characterization of the LST physical and chemical properties. Furthermore, batch adsorption experiments demonstrated that LST had superior adsorption property and adsorption selectivity toward cationic dyes compared to those of anionic dyes. A multifarious influencing effect on the cationic dye adsorption behavior during the adsorption process was systematically investigated. Moreover, the pseudo-second-order kinetic model felicitously depicted the cationic dye adsorption behavior through an elaborate kinetic study, namely, chemisorption was the main adsorption action. Meanwhile, different adsorption isotherm models were utilized to process the experimental data, uncovering that the adsorption isotherms of cationic dyes on LST were suitable for a Langmuir isothermal model. More importantly, an ion-exchange mechanism was proposed for the cationic dye adsorption on LST, and the ion-exchange reaction occurred with a stoichiometric exchange between 1 mol of Na+ ions in the LST interlayer and 1 mol of MB molecules in the solution. In parallel, the electrochemical impedance spectroscopy and cyclic voltammogram measurements verified that the high ionic conductivity of Na+ ions in the LST interlayer resulted in a superior adsorption property. A two-step acid-base procedure was ultimately adopted to effectively regenerate LST adsorbent. This work provides not only an alternative adsorbent with superior adsorption capacity and adsorption selectivity but also some guiding significance for research on the adsorption mechanism of layered titanates.
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Nanopores are powerful single-molecule tools for label-free sensing of nanoscale molecules including DNA that can be used for building designed nanostructures and performing computations. Here, DNA hard drives (DNA-HDs) are introduced based on DNA nanotechnology and nanopore sensing as a rewritable molecular memory system, allowing for storing, operating, and reading data in the changeable three-dimensional structure of DNA. Writing and erasing data are significantly improved compared to previous molecular storage systems by employing controllable attachment and removal of molecules on a long double-stranded DNA. Data reading is achieved by detecting the single molecules at the millisecond time scale using nanopores. The DNA-HD also ensures secure data storage where the data can only be read after providing the correct physical molecular keys. Our approach allows for easy-writing and easy-reading, rewritable, and secure data storage toward a promising miniature scale integration for molecular data storage and computation.
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ADN , Almacenamiento y Recuperación de la Información , Nanoporos , ADN/genética , NanotecnologíaRESUMEN
Anion binding by receptor molecules is a central field of modern chemistry which impacts areas of catalysis as well as biological and materials chemistry. As binding often requires high chemical stability under aerobic and aqueous conditions for practical applications, carbon-based anion receptors have dominated this field, with main group element analogues receiving far less attention. The recent observation that the air- and moisture-stable amino-cyclophosph(V)azanes of the type [RN(E)P(µ-NR)]2 (E = O, S, Se) can exhibit halide binding that is competitive with topologically related organic receptors (such as squaramides and thioureas) has motivated us here to explore how the binding properties of phosphazane receptors can be enhanced further. Coordination of transition metals by the two P,N metal coordination sites of the phosph(III)azane dimer [(2-py)NHP(µ-NtBu)]2 not only activates the receptor for anion binding (by fixing the optimum exo-exo conformation and polarizing the endocyclic N-H substituents) but also stabilizes the P2N2 ring to hydrolysis and oxidation. We show how the binding properties of these receptors can be modulated by the coordinated metal fragments and that they can bind chloride 1 to 2 orders of magnitude stronger than the related squaramides and thioureas. These features can be utilized in anion transport through phospholipid bilayers under aqueous conditions for which transport can be improved by 1 order of magnitude compared to the previous best phosphazane and thiourea transporters. This study demonstrates how careful design of inorganic systems can result in potent supramolecular functionality, beyond that observed for organic counterparts.
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A series of samples including leaf-like and rod-like rutile TiO2 nanoparticles with various facets exposed on the surface, parallelepiped-shaped anatase nanoparticles with [111] vertical facet exposed on the surface, irregular anatase nanoparticles, microsized six-point star-like anatase aggregates, and almond-like brookite aggregates had been hydrothermally synthesized from lepidocrocite-type layered titanate nanosheets. A systematical investigation was established to uncover the phase transition and morphological evolution from nanosheets to TiO2 polymorphs, and a phase diagram was determined by adjusting the synthesis parameters of the pH value and temperature. Two kinds of mechanisms composed of the dissolution-deposition process following Ostwald's ripening mechanism and the in situ topochemical conversion process following Ostwald's step rule had been proposed based on the time-dependent hydrothermal experiments. Briefly, the formation of the single-crystalline rutile phase appeared only at high temperatures with very low pH values, and similarly, the brookite phase strictly formed at high temperatures with a very high pH value. Nevertheless, the anatase phase could moderately appear in a wide range of temperatures and pH values. In addition, the single-crystalline rutile adopted a leaf-like morphology at low temperatures with high pH values and a rod-like morphology at high temperatures with low pH values, while the morphological evolution of anatase particles proceeded from irregular to parallelepiped-shaped and finally to six-point star-like morphology, and the crystal size was reduced from 1000 to 5 nm with decreasing pH values. Meanwhile, with the prolongation of the hydrothermal time, the layered titanate nanosheets first dissolved into the amorphous state and further converted into small anatase nanoparticles and finally to rutile or anatase nanoparticles based on the dissolution-deposition process, or the {010}-faceted layered titanate structure first converted into the [111]-vertical faceted anatase nanosheets by the topochemical transformation reaction and then split into the [111]-vertical faceted anatase nanoparticles. More importantly, the mesoporous [111]-vertical faceted anatase nanoparticles exhibited enhanced photocatalytic performance compared to that of Degussa P25, which was ascribed to its superior electronic band structure and effective charge separation. The systematical investigation in this work would be significant for consummating the preparation of the TiO2 polymorphs from layered titanate nanosheets and provided some reference values and guide schemes for the preparation of TiO2 nanoparticles with outstanding photocatalytic performance.
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G-quadruplexes (Gqs) are guanine-rich DNA structures formed by single-stranded DNA. They are of paramount significance to gene expression regulation, but also drug targets for cancer and human viruses. Current ensemble and single-molecule methods require fluorescent labels, which can affect Gq folding kinetics. Here we introduce, a single-molecule Gq nanopore assay (smGNA) to detect Gqs and kinetics of Gq formation. We use â¼5 nm solid-state nanopores to detect various Gq structural variants attached to designed DNA carriers. Gqs can be identified by localizing their positions along designed DNA carriers, establishing smGNA as a tool for Gq mapping. In addition, smGNA allows for discrimination of (un)folded Gq structures, provides insights into single-molecule kinetics of Gq folding, and probes quadruplex-to-duplex structural transitions. smGNA can elucidate the formation of Gqs at the single-molecule level without labeling and has potential implications on the study of these structures both in single-stranded DNA and in genomic samples.
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ADN de Cadena Simple/química , G-Cuádruplex , Nanoporos/ultraestructura , ADN/química , Cinética , Nanotecnología , Conformación de Ácido Nucleico , Imagen Individual de MoléculaRESUMEN
Solid-state nanopores are powerful tools for reading the three-dimensional shape of molecules, allowing for the translation of molecular structure information into electric signals. Here, we show a high-resolution integrated nanopore system for identifying DNA nanostructures that has the capability of distinguishing attached short DNA hairpins with only a stem length difference of 8 bp along a DNA double strand named the DNA carrier. Using our platform, we can read up to 112 DNA hairpins with a separating distance of 114 bp attached on a DNA carrier that carries digital information. Our encoding strategy allows for the creation of a library of molecules with a size of up to 5 × 1033 (2112) that is only built from a few hundred types of base molecules for data storage and has the potential to be extended by linking multiple DNA carriers. Our platform provides a nanopore- and DNA nanostructure-based data storage method with convenient access and the potential for miniature-scale integration.
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ADN/química , Almacenamiento y Recuperación de la Información/métodos , Nanoporos , Nanoestructuras/química , Nanotecnología/métodos , Secuencia de Bases , Electricidad , Biblioteca de Genes , Nanoporos/ultraestructura , Nanoestructuras/ultraestructuraRESUMEN
A water-soluble self-assembled supramolecular FeII4L4 tetrahedron binds to single stranded DNA, mismatched DNA base pairs, and three-way DNA junctions. Binding of the coordination cage quenches fluorescent labels on the DNA strand, which provides an optical means to detect the interaction and allows the position of the binding site to be gauged with respect to the fluorescent label. Utilizing the quenching and binding properties of the coordination cage, we developed a simple and rapid detection method based on fluorescence quenching to detect unpaired bases in double-stranded DNA.
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Disparidad de Par Base , ADN de Cadena Simple/metabolismo , Compuestos Ferrosos/química , Compuestos Ferrosos/metabolismo , ADN/química , ADN/metabolismo , ADN de Cadena Simple/química , FluorescenciaRESUMEN
The binding and sensing of anions is an important cross-disciplinary field, which impacts broad areas such as biology, supramolecular chemistry and catalysis. To date, however, this area has been dominated by organic architectures which function as H-bonding, anion receptor molecules. Inorganic anion receptors have largely been based on Lewis acidic metals, with very few examples of H-bonding counterparts of organic systems having been systematically studied. This paper develops strategies for enhancing the anion binding properties of phosphazanes of the type [(RNH)(E)P(µ-N tBu)]2 (E = O, S, Se) which are bench-stable, H-bond receptors that can be regarded as inorganic analogues of squaramides (a key class of organic anion receptor). The distinct advantages of these inorganic receptors over organic counterparts is the ease by which their functionality and electronic character can be altered (by means of the R group, chalcogenide, or metal present). Se substitution at the P centers, the presence of electron-withdrawing R groups, and metal coordination to the soft donor centers can be used to modulate and enhance anion binding. The water stability and superior anion binding properties of the seleno-phosph(V)azanes give them applications as synthetic anion transporters through phospholipid layers.
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Compuestos Organofosforados/química , Enlace de Hidrógeno , Ligandos , Metales/química , Modelos Moleculares , Conformación Molecular , Selenio/químicaRESUMEN
OBJECTIVE: To evaluate the efficacy and safety of early unclamping laparoscopic partial nephrectomy (LPN) for complex renal tumor relative to the standard artery clamping technique (SCT). METHODS: Sixty-one patients with complex renal tumor (RENAL score ≥7) underwent LPN at our institution from January 2013 to April 2017. LPN was performed via SCT in 32 patients and via the early unclamping technique (EUT) in 29 patients. Operation time, warm ischemia time (WIT), blood loss, bleeding requiring transfusion, tumor volume, excisional volume loss (EVL), complications, and renal function before and after operation of the affected kidney were compared between the groups. RESULTS: All surgeries were successful without conversion to open or nephrectomy. EUT reduced the WIT (p < 0.001) but did not increase the complication rate (p = 0.322). Although the tumor volume and EVL were larger in the EUT than in the SCT group (p = 0.011, p = 0.001), glomerular filtration rate (GFR) reduction in the affected kidney did not significantly differ between the groups (p = 0.120). CONCLUSION: Early unclamping LPN for complex renal tumor is safe and efficient. Additionally, the EUT could expand the application of LPN in complex renal tumors, and make this challenging surgery easier and safer.
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Neoplasias Renales/cirugía , Laparoscopía/métodos , Nefrectomía/métodos , Isquemia Tibia , Adulto , Anciano , Pérdida de Sangre Quirúrgica , China , Femenino , Estudios de Seguimiento , Tasa de Filtración Glomerular , Humanos , Riñón/patología , Masculino , Persona de Mediana Edad , Tempo Operativo , Seguridad del Paciente , Estudios Retrospectivos , Robótica , Resultado del TratamientoRESUMEN
Metal-organic anion channels based on Zn10 L15 pentagonal prisms have been prepared by subcomponent self-assembly. The insertion of these prisms into lipid membranes was investigated by ion-current and fluorescence measurements. The channels were found to mediate the transport of Cl- anions through planar lipid bilayers and into vesicles. Tosylate anions were observed to bind and plug the central channels of the prisms in the solid state and in solution. In membranes, dodecyl sulfate blocked chloride transport through the central channel. Our Zn10 L15 prism thus inserts into lipid bilayers to turn on anion transport, which can then be turned off through addition of the blocker dodecyl sulfate.
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Aqueous N-rich carbon dots (CDs), prepared by the microwave-assisted pyrolysis method, are applied as a dual sensing platform for both the fluorescent and electrochemical detection of 2,4,6-trinitrotoluene (TNT). The fluorescent sensing platform is established on the strong TNT-amino interaction which can quench the photoluminescence of amino functionalized CDs through charge transfer. The resultant linear detection ranges from 10 nM to 1.5 µM with a fast response time of 30 s. Glassy carbon electrode modified with CDs exhibits a fine capability for TNT reduction with the linear range from 5 nM to 30 µM, better than that obtained by the fluorescent method. Moreover, the minimum distinguishable response concentration with respect to these two methods is down to the nanomolar level with a high specificity and sensitivity.