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A fast PCR-assisted impedimetric biosensor was developed for the selective detection of the clbN gene from the polyketide synthase (pks) genomic island in real Escherichia coli samples. This genomic island is responsible for the production of colibactin, a harmful genotoxin that has been associated with colorectal cancer. The experimental protocol consisted of immobilizing the designated forward primer onto an Au electrode surface to create the sensing probe, followed by PCR temperature cycling in blank, positive, and negative DNA controls. Target DNA identification was possible by monitoring changes in the system's charge transfer resistance values (Rct) before and after PCR treatment through electrochemical impedance spectroscopy (EIS) analysis. Custom-made, flexible gold electrodes were fabricated using chemical etching optical lithography. A PCR cycle study determined the optimum conditions to be at 6 cycles providing fast results while maintaining a good sensitivity. EIS data for the DNA recognition process demonstrated the successful distinction between target interaction resulting in an increase in resistance to charge transfer (Rct) percentage change of 176% for the positive DNA control vs. 21% and 20% for the negative and non-DNA-containing controls, respectively. Results showed effective fabrication of a fast, PCR-based electrochemical biosensor for the detection of pks genomic island with a calculated limit of detection of 17 ng/µL.
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Técnicas Biossensoriais/métodos , Espectroscopia Dielétrica/métodos , Escherichia coli/genética , Genoma Bacteriano , Peptídeos/genética , Policetídeo Sintases/genética , Reação em Cadeia da Polimerase/métodos , Limite de Detecção , PolicetídeosRESUMO
In this study we probe the electrocatalytic activity of Pt nanoparticles supported on ceria nanoparticles (NPs) and nanorods (NRs) in the ethanol oxidation reaction (EOR) in alkaline media. The goal of this study was to relate morphology, support structure and composition to the EOR catalytic activity by using in situ X-ray absorption fine structure (XAFS) studies. Cyclic voltammetry experiments showed that both ceria supported catalysts (NP vs. NR) had similar peak current densities at fast scan rates, however at slow scan rates, the ceria NR catalyst showed superior catalytic activity. In situ XAFS studies in KOH showed that both ceria supported catalysts had more electron density in their d-band (with the ceria NR having more electron density overall) than ceria - free Pt/Vulcan standard. However, in an ethanol solution the ceria NR catalyst had the least electron density. We propose that this change is due to the increased charge transfer efficiency between the ceria nanorod support and platinum. In the KOH solution, the increased electron density makes the platinum less electrophilic and hinders Pt-OH bond formation. In the EtOH solution, platinum's increased nucleophilicity facilitates the bond formation between Pt and the electron deficient carbon in ethanol which in turn withdraws the electron density from platinum and increases the white line intensity as observed in the XAS measurements.
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We combined various modalities of the optical-optical double resonance (OODR) photoionization technique to simplify the interpretation of crowded molecular spectra. To demonstrate the effectiveness of our method, we applied it to the 64000 to 65200 cm(-1) spectral region of the molecule NO, where exist the following electronic states: B (2)Π (v = 21), D (2)Σ(+) (v = 5), F (2)Δ (v = 1), L (2)Π (v = 3), and K (2)Π (v = 0). This spectral region is complicated because (1) several electronic states are close in energy, (2) some of the rotational energy patterns are irregular, and (3) the relative intensity of the different bands varies markedly. We implemented four modalities of the OODR experimental technique that involved the combined use of two or three lasers. The individual rotational levels up to N' = 20 of the A(2)Σ(+) (v = 0) state were pumped as intermediate states by one-photon excitation from appropriate rotational levels in the X(2)Π (v = 0) ground state. Some of the schemes implemented provided information about line positions and relative band intensities, whereas the ion-dip detection scheme provided insight into the fate of the population in the different states. The term values that we derived are in good agreement with the literature ones. We rotationally resolved the spectra for the K (2)Π (v = 0) and B (2)Π (v = 21) states up to N = 20, and for the D (2)Σ(+) (v = 5) and L (2)Π (v = 3) states up to N = 8 and 7, respectively. Strangely, only in the rotational levels between N = 6 and N = 20 were we able to observe the F (2)Δ state, which is mostly mixed with the B' (2)Δ (v = 4) state and usually notated as F (2)Δ (v = 1) â B' (2)Δ (v = 4). We obtained the rotational constants for the B (2)Π1/2 (v = 21), L (2)Π3/2 (v = 3), and K (2)Π1/2 (v = 0) states, which had not been previously reported.
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Lasers , Óxido Nítrico/química , Fótons , Análise EspectralRESUMO
BACKGROUND: This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO. RESULTS: Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation. CONCLUSIONS: It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.
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Fenômenos Biofísicos , Grafite/química , Óxidos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Biocatálise , Caproatos/química , Dicroísmo Circular , Medição da Troca de Deutério , Dextranos/química , Glicosilação , Proteínas Imobilizadas/metabolismo , Cinética , Nanopartículas/química , Oxirredução , Espectroscopia Fotoeletrônica , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , TemperaturaRESUMO
Nanotechnology allows the synthesis of nanoscale catalysts, which offer an efficient alternative for fuel cell applications. In this laboratory experiment, the student selects a cost-effective anode for fuel cells by comparing three different working electrodes. These are commercially available palladium (Pd) and glassy carbon (GC) electrodes, and a carbon paste (CP) electrode that is prepared by the students in the laboratory. The GC and CP were modified with palladium nanoparticles (PdNP) suspensions. The electrodes efficiencies were studied for ethanol oxidation in alkaline solution using cyclic voltammetry techniques. The ethanol oxidation currents obtained were used to determine the current density using the geometric and surface area of each electrode. Finally, students were able to choose the best electrode and relate catalytic activity to surface area for ethanol oxidation in alkaline solution by completing a critical analysis of the cyclic voltammetry results. With this activity, fundamental electrochemical concepts were reinforced.
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The synthesis of shape-controlled nanoparticles is currently a hot research topic. However, from an applied point of view, there is still a lack of easy, cheap, and scalable methodologies. In this communication we report, for the first time, the synthesis of cubic platinum nanoparticles with a very high yield using a water-in-oil microemulsion method, which unlike others, such as the colloidal method, fulfills the previous requirements. This shape/surface structure control is determined by the concentration of HCl in the water phase of the microemulsion. The results reported here show that the optimal HCl percentage in the water phase is about 25% to obtain the highest amount of cubic nanostructures. Ammonia electro-oxidation is used as a surface structure sensitive reaction to illustrate HCl surface structure effects. Moreover, in situ electrochemical characterization has been performed to study the nanoparticle surface structure.
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The increasing number of applications for shape-controlled metal nanoparticles (NPs) has led to the need for easy, cheap, and scalable methodologies. We report the synthesis of (100) preferentially oriented Pt NPs, with a particle size of 9 nm, by using a water-in-oil microemulsion method. The specific surface structure of the NPs is induced by the presence of H(2)SO(4) in the water phase of the microemulsion. Interestingly, the results reported herein show how increasing amounts of H(2)SO(4) lead to the formation of Pt NPs containing a larger amount of (100) sites on their surface. This preferential surface orientation is confirmed electrochemically by using the so-called hydrogen adsorption/desorption process. In addition, transmission electron microscopy measurements confirm the presence of cubic-like Pt NPs. Finally, the electrocatalytic properties of the Pt NPs are evaluated towards ammonia and CO electro-oxidations, which are (100) structure-sensitive reactions.
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An electrodeposition technique of low-enriched uranium onto boron-doped diamond (BDD) electrodes for uranium electro-assembling, sequestration, uranium electrowinning (as the electroextraction alternative), and future neutron detection applications has been developed. Our findings through physicochemical characterization and an in-depth XPS analysis show that the U/BDD system consists of a blend of uranium oxides with IV, V, and VI oxidation states. Results show that U5+ is present and stable under open atmospheric conditions. The U electrodeposition on BDD creates smooth surfaces, free of voids, with uniform deposition of homogeneous tiny particles of stable uranium oxides, instead of chunky particles, and uranium compound mixtures, like large fibers of the precursor uranyl. Our electrochemical method operates without high temperatures or hazardous compounds. Uranium corrosion and oxidation processes occur spontaneously and parallel to the electrochemical formation of metallic uranium on BDD electrode surfaces, with metallic uranium reacting with water, producing fine particles of UO2. This work represents the first attempt to create a surface of uranium oxides, where the film thickness can be controlled for future applications, e.g., improving sensitivity in neutron detection technologies. Our U electro-assembling method provides a sustainable strategy for uranium electro-recovery from nuclear wastes, immobilizing uranium as a storage method or as U-film fabrication (U/BDD) for future neutron detection applications. Besides, this work contributes to uranium-based technologies, improving them and providing a better understanding of their electrochemical properties, e.g., uranium redox processes, uranium oxides' formation, and stability evaluation. These properties are of remarkable need for uranium-based target formation. The use of our U/BDD method is proposed as an environmental protocol to recover and immobilize uranium-235, and other fissile materials, from civil and defense wastes, contaminated systems, and stockpiles.
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Designing N-coordinated porous single-atom catalysts (SACs) for the oxygen reduction reaction (ORR) is a promising approach to achieve enhanced energy conversion due to maximized atom utilization and higher activity. Here, we report two Co(II)-porphyrin/ [2,1,3]-benzothiadiazole (BTD)-based covalent organic frameworks (COFs; Co@rhm-PorBTD and Co@sql-PorBTD), which are efficient SAC systems for O2 electrocatalysis (ORR). Experimental results demonstrate that these two COFs outperform the mass activity (at 0.85 V) of commercial Pt/C (20%) by 5.8 times (Co@rhm-PorBTD) and 1.3 times (Co@sql-PorBTD), respectively. The specific activities of Co@rhm-PorBTD and Co@sql-PorBTD were found to be 10 times and 2.5 times larger than that of Pt/C, respectively. These COFs also exhibit larger power density and recycling stability in Zn-air batteries compared with a Pt/C-based air cathode. A theoretical analysis demonstrates that the combination of Co-porphyrin with two different BTD ligands affords two crystalline porous electrocatalysts having different d-band center positions, which leads to reactivity differences toward alkaline ORR. The strategy, design, and electrochemical performance of these two COFs offer a pyrolysis-free bottom-up approach that avoids the creation of random atomic sites, significant metal aggregation, or unpredictable structural features.
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An autonomous electrochemical system prototype for ammonia oxidation reaction (AOR) measurements was efficiently done inside a 4'' x 4'' x 8'' 2U Nanoracks module at the International Space Station (ISS). This device, the Ammonia Electrooxidation Lab at the ISS (AELISS), included an autonomous electrochemical system that complied with NASA ISS nondisclosure agreements, power, safety, security, size constrain, and material compatibility established for space missions. The integrated autonomous electrochemical system was tested on-ground and deployed to the International Space Station as a "proof-of-concept" ammonia oxidation reaction testing space device. Here are discussed the results of cyclic voltammetry and chronoamperometry measurements done at the ISS with a commercially available channel flow-cell with eight screen-printed electrodes, including Ag quasi-reference (Ag QRE) and carbon counter electrodes. Pt nanocubes in Carbon Vulcan XC-72R were used as the catalyst for the AOR and 2 µL drop of Pt nanocubes/ Carbon Vulcan XC-72R, 20 wt%, ink was placed on the carbon working electrodes and allowed to dry in air. After the AELISS was prepared for launch to the ISS, a 4 days delayed (2 days in the space vehicle Antares and 2 days space transit to the ISS) cause a slight shift on the Ag QRE potential. Nevertheless, the AOR cyclic voltametric peak was observed in the ISS and showed ca. 70% current density decrease due to the buoyancy effect in agreement with previous microgravity experiments done at the zero-g aircraft.
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An effort to develop smaller, well-dispersed catalytic materials electrochemically on high-surface-area carbon supports is required for improved fuel cell performance. A high-surface-area carbon material of interest is carbon nano-onions (CNOs), also known as multilayer fullerenes. The most convenient synthesis method for CNOs is annealing nanodiamond particles, thus retaining the size of the precursors and providing the possibility to prepare very small nanocatalysts using electrochemical techniques. In terms of pure metal catalysts, platinum is the most common catalyst used in fuel cells. The combination of Pt nanoparticles with CNOs could lead to new catalytic nanomaterials. In this work, this was accomplished by using a rotating disk-slurry electrode (RoDSE) technique. The Pt/CNO catalysts were prepared from slurries that contained functionalized CNOs and K(2)PtCl(6) as the platinum precursor in aqueous 0.1 M H(2)SO(4) solution. X-ray photoelectron spectroscopy results showed that 37% of the Pt on the CNOs is metallic Pt whereas 63% had higher binding energies, which is evidence of higher oxidation states or the presence of Pt atoms and clusters on CNOs. However, aberration-corrected scanning transmission electron microscopy of the Pt/CNOs confirmed the presence of Pt atoms and clusters on CNOs. Thermal gravimetric analysis showed the excellent thermal stability of the Pt/CNOs and a lower onset potential for the electrochemical oxidation of methanol compared to that of commercial Pt/Vulcan catalyst material. The computational method confirmed the Pt atoms' location at CNOs surface sites. Geometric parameters for distances between Pt atoms in the 3Pt/CNOs molecular system from our theoretical calculations are in agreement with the respective parameters obtained experimentally. The combination of CNO with RoDSE presents a new highly dispersed catalyst nanomaterial.
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Fulerenos/química , Nanoestruturas/química , Platina/química , Catálise , Eletrodos , Galvanoplastia , Metanol/química , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Nanoestruturas/ultraestrutura , Oxirredução , Tamanho da Partícula , Espectroscopia Fotoeletrônica , Propriedades de Superfície , TermodinâmicaRESUMO
This paper reports the decoration of single wall carbon nanotubes (SWCNTs) with platinum (Pt) nanoparticles using an electrochemical technique, rotating disk slurry electrode (RoDSE). Pt/SWCNTs were electrochemically characterized by cyclic voltammetry technique (CV) and physically characterized through the use of transmission electron microscopy (TEM), energy dispersive spectroscopy - X-ray florescence (EDS-XRF) and X-ray diffraction (XRD). After characterization it was found that electrodeposited nanoparticles had an average particle size of 4.1 ± 0.8 nm. Pt/SWCNTs were used as sensing material for methane (CH4) detection and showed improved sensing properties in a range of concentration from 50 ppm to 200 ppm parts per million (ppm) at room temperature, when compared to other Pt/CNTs-based sensors. The use of this technique for the preparation of Pt/SWCNTs opens a new possibility in the bulk preparation of samples using an electrochemical method and thus their potential use in a wide variety of applications in chemical sensing, fuel cell and others.
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Telomerase overexpression has been associated directly with cancer, and the enzyme itself is recognized within the scientific community as a cancer biomarker. BIDEA's biosensing strip (BBS) is an innovative technology capable of detecting the presence of telomerase activity (TA) using electrochemical impedance spectroscopy (EIS). This BBS is an interdigital gold (GID) electrode array similar in size and handling to a portable glucose sensor. For the detection of the biomarker, BBS was modified by the immobilization of a telomere-like single strand DNA (ssDNA) on its surface. The sensor was exposed to telomerase-positive extract from commercially available cancer cells, and the EIS spectra were measured. Telomerase recognizes the sequence of this immobilized ssDNA probe on the BBS, and the reverse transcription process that occurs in cancer cells is replicated, resulting in the ssDNA probe elongation. This surface process caused by the presence of TA generates changes in the capacitive process on the electrode array microchip surface, which is followed by EIS as the sensing tool and correlated with the presence of cancer cells. The telomerases' total cell extraction protocol results demonstrate significant changes in the charge-transfer resistance (R ct) change rate after exposure to telomerase-positive extract with a detection limit of 2.94 × 104 cells/mL. Finally, a preliminary study with a small set of "blind" uterine biopsy samples suggests the feasibility of using the changes in the R ct magnitude change rate (Δ(ΔR ct/R cti)/Δt) to distinguish positive from negative endometrial adenocarcinoma samples by the presence or absence of TA.
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Isomeric tetraphenylbenzodifuran systems, benzo[1,2-b:5,4]difuran and benzo[1,2-b:4,5]difuran, containing electron acceptor groups (CF(3), CN, and NO(2)) have been synthesized and studied. Their electronic absorption, fluorescence, two-photon absorption cross sections, and electrochemical properties were investigated. The absorption and emission maxima are red-shifted for the linear-conjugated systems in comparison with the corresponding isomer. Dual fluorescence was observed and the existence of a twisted intramolecular charge transfer state was confirmed by low-temperature emission experiments. Wide HOMO-LUMO energy gaps were obtained ranging from 2.53 to 3.28 eV. HOMO levels were found in the energy range of -6.03 to -6.63 eV while LUMO are within -2.55 to -3.52 eV.
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Benzofuranos/química , Benzofuranos/síntese química , Eletroquímica , Fluorescência , Estrutura Molecular , Teoria Quântica , EstereoisomerismoRESUMO
Highly dispersed iron-based quantum dots (QDs) onto powdered Vulcan XC-72R substrate were successfully electrodeposited by the rotating disk slurry electrodeposition (RoDSE) technique. Our findings through chemical physics characterization revealed that the continuous electron pathway interaction between the interface metal-carbon is controlled. The rotating ring-disk electrode (RRDE) and the prototype generation unit (PGU) of in-situ H2O2 generation in fuel cell experiments revealed a high activity for the oxygen reduction reaction (ORR) via two-electron pathway. These results establish the Fe/Vulcan catalyst at a competitive level for space and terrestrial new materials carriers, specifically for the in-situ H2O2 production. Transmission electron microscopy (TEM) analysis reveals the well-dispersed Fe-based quantum dots with a particle size of 4 nm. The structural and chemical-physical characterization through induced coupled plasma-optical emission spectroscopy (ICP-OES), transmission scanning electron microscopy (STEM), X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and X-ray absorption spectroscopy (XAS); reveals that, under atmospheric conditions, our quantum dots system is a Fe2+/3+/Fe3+ combination. The QDs oxidation state tunability was showed by the applied potential. The obtention of H2O2 under the compatibility conditions of the drinking water resources available in the International Space Station (ISS) enhances the applicability of this iron- and carbon-based materials for in-situ H2O2 production in future space scenarios. Terrestrial and space abundance of iron and carbon, combined with its low toxicity and high stability, consolidates this present work to be further extended for the large-scale production of Fe-based nanoparticles for several applications.
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Onion-like carbon nanoparticles were synthesized from diamond nanoparticles to be used as the precursor for graphene oxide quantum dots. Onion-like carbon nanoparticles were exfoliated to produce two types of nanoparticles, graphene oxide quantum dots that showed size-dependent fluorescence and highly stable inner cores. Multicolor fluorescent quantum dots were obtained and characterized using different techniques. Polyacrylamide gel electrophoresis showed a range of emission wavelengths spanning from red to blue with the highest intensity shown by green fluorescence. Using high-resolution transmission electron microscopy, we calculated a unit cell size of 2.47 Å in a highly oxidized and defected structure of graphene oxide. A diameter of ca. 4 nm and radius of gyration of ca. 11 Å were calculated using small-angle X-ray scattering. Finally, the change in fluorescence of the quantum dots was studied when single-stranded DNA that is recognized by telomerase was attached to the quantum dots. Their interaction with the telomerase present in cancer cells was observed and a change was seen after six days, providing an important application of these modified graphene oxide quantum dots for cancer sensing.
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Studying the oxygen reduction reaction (ORR) in the alkaline electrolyte has proven to promote better catalytic responses and accessibility to commercialization. Ni-nanowires (NWs) were synthesized via the solvothermal method and modified with Pt using the spontaneous galvanic displacement method to obtain PtNi-NWs. Carbon Vulcan XC-72R (V) was used as the catalyst support, and they were doped with NH3 to obtain PtNi-NWs/V and PtNi-NWs/V-NH3. Their electrocatalytic response for the ORR was tested and PtNi-NWs/V provided the highest specific activity with logarithmic values of 0.707 and 1.01 (mA/cm2 Pt) at 0.90 and 0.85 V versus reversible hydrogen electrode (RHE), respectively. PtNi-NWs showed the highest half-wave potential (E 1/2 = 0.89 V) at 1600 rpm and 12 µgPt/cm2 in 0.1 M KOH at 25.00 ± 0.01 °C. Additionally, the catalysts followed a four-electron pathway according to the Koutecký-Levich analysis. Moreover, durability experiments demonstrated that the PtNi-NW/V performance loss was like that of commercial Pt/V along 10,000 cycles. Electrochemical ORR in situ X-ray absorption spectroscopy results showed that the Pt L3 edge white line in the PtNi-NW catalysts changed while the electrochemical potential was lowered to negatives values, from 1.0 to 0.3 V versus RHE. The Pt/O region in the in situ Fourier transforms remained the same as the potentials were applied, suggesting an alloy formation between Pt and Ni, and Pt/Pt contracted in the presence of Ni. These results provide a better understanding of PtNi-NWs in alkaline electrolytes, suggesting that they are active catalysts for ORR and can be tuned for fuel cell studies.
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A density-functional theory study of the electrochemical adsorption of sulfuric acid anions was conducted at the Pt(111)/electrolyte interface over a wide range of electrode potential, including the anomalous region of the hydrogen voltammogram of this electrode. We focus on the precise nature of the binding species and their bonding to the surface, identifying the adsorbed species as a function of electrode potential. In particular, the origin of anomalous or so-called "butterfly" feature in this voltammogram between +0.30 and +0.50 V vs. the reference hydrogen electrode and the nature of the adsorbed species on the Pt(111) surface in this potential range were explicated.
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The optical-optical double resonance time of flight (OODR-TOF) spectroscopy technique was employed to examine the 65,000-66,500 cm(-1) region of the nitric oxide spectrum. In this region, we detected the following three electronic states: E (2)Sigma(+) (nu = 2) (Rydberg state), B (2)Pi (nu = 23) (valence state), and L (2)Pi (nu = 4) (valence state). The rotational structure analysis of an unexpected band in the red part of the spectra revealed the presence of a new super-excited (2)Sigma(+) Rydberg state at approximately 13.3 eV, which was populated through a three-photon transition from the intermediate A (2)Sigma(+) (nu = 0) state. This super-excited state converges to the NO (a(3)Sigma(+)) ionic state with electronic configuration (1sigma)(2)(2sigma)(2)(3sigma)(2)(4sigma)(2)(5sigma)(2)(1pi)(3)(2pi)(1)(3ssigma)(1).
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Lasers , Óxido Nítrico/química , Fótons , Fenômenos Químicos , Elétrons , Análise EspectralRESUMO
Carbon nanotubes (CNTs) have been broadly studied due to their exceptional structural, electronic and mechanical properties, and their use have been proposed for many applications. The number of biosensing applications of CNTs has increased in the past few years. Nevertheless, in order to use CNTs as standard materials in the biosensing field and to take full advantage of their unique properties, several problems must be solved. For example, the solubility of CNTs, especially in aqueous solvents, needs to be improved. Furthermore, reliable methodologies for the easy, reproducible and efficient incorporation of CNTs into solid substrates must be developed. Chemical functionalization of CNTs with biomolecules, and specifically with DNA, provides an alternative to the previous challenges. This review will present a brief overview of the methods available for the chemical functionalization of CNTs with DNA by either covalent or non-covalent means. Moreover, the main scope will be on describing the use of DNA-CNT complexes in biosensing applications. Detection of ions, glucose, peroxide and DNA hybridization using the DNA-CNT hybrids will be discussed.