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The digestive characteristics of wheat starch are closely related to human health. However, the digestive mechanisms of distinct wheat starch granules are not well understood. To address this problem, A- and B-type wheat starch granules (AWS and BWS, respectively) were digested in vitro and the structural evolution of the digestive remnants was compared. After stomach-intestinal digestion of AWS, its crystallinity decreased from 12.75â¯% to 6.65â¯%, its fractal dimension decreased from 3.12 to 2.35, and the median particle size decreased from 20.613 to 10.135⯵m. Additionally, the number of short chains (polymerization degree<14) and thermodynamic stability decreased after digestion. For BWS, Fourier transform infrared ratio of 1047/1022â¯cm-1 and 995/1022â¯cm-1 increased from 0.665 and 0.725 to 0.990 and 0.800, respectively. The median particle size decreased from 5.480 to 4.769⯵m. An enzyme-resistant scattering peak was observed in the 1.35â¯nm-1 lamellar structure. Additionally, the number of B2 and B3 chains and the thermodynamic stability increased after digestion. Our study confirmed that BWS is more likely than AWS to form enzyme-resistant structures during digestion. These findings provide insights into the distinct digestion mechanisms of AWS and BWS, and serve as a foundation for modifying wheat starch to increase its nutritional value.
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This study aimed to explore the potential antioxidant activity and mechanism of oligopeptides from sauce-aroma Baijiu. The oligopeptides of Val-Leu-Pro-Phe (VLPF), Pro-Leu-Phe (PLF), Val-Gly-Phe-Cys (VGFC), Leu-Tyr-Pro (LYP), Leu-Pro-Phe (LPF), and Phe-Thr-Phe (FTF) were identified by liquid chromatography-mass spectrometry (LC-MS) from the mixed-distillate of Baijiu fermented grains and soy sauce residue (MDFS). The antioxidant mechanism of these oligopeptides on scavenging DPPHâ¢, ABTSâ¢+, and hydroxide radicals was investigated, respectively. Among them, VGFC had the strongest potential antioxidant activity, which was responsible for its hydrogen bonds with these radicals with high affinity. The binding energies between VGFC and these radicals were -1.26 kcal/mol, -1.33 kcal/mol, and -1.93 kcal/mol, respectively. Additionally, free radicals prefer to bind the oligopeptide composed of hydrophobic amino acid residues such as Leu, Val, Phe, and Pro, thus being scavenged for exerting antioxidant activity. It provided a new idea for the development and utilization of bioactive oligopeptides in sauce-aroma Baijiu.
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INTRODUCTION: Previously reported preparation methods of Ginkgo biloba leaf extract (EGBL) have mainly focused on the enrichment of flavonoid glycosides (FG) and terpene trilactones (TT), which led to the underutilization of G. biloba leaves (GBL). OBJECTIVES: To make full use of GBL, in this study, a comprehensive optimization strategy for preparing EGBL by macroporous resin column chromatography was proposed and applied to enrich FG, TT, and shikimic acid (SA) from GBL. METHODOLOGY: Initially, the static adsorption and desorption were executed to select suitable resin. Then, the influences of solution pH were investigated by the static and dynamic adsorption. Subsequently, eight process parameters were systematically investigated via a definitive screening design (DSD). After verification experiments, scale-up enrichment was carried out, investigating the feasibility of the developed strategy for application on an industrial scale. RESULTS: It was found that XDA1 was the most appropriate adsorbent for the preparation of EGBL at solution pH 2.0. Furthermore, based on the constraints of the desired quality attributes, the optimized ranges of operating parameters were successfully acquired, and the verification experiments demonstrated the accuracy and reliability of using DSD to investigate the chromatography process for the preparation of EGBL. Finally, magnified experiments were successfully performed, obtaining the EGBL containing 26.54% FG, 8.96% TT, and 10.70% SA, which reached the SA level of EGB761, an international standard EGBL. CONCLUSION: The present study not only provided an efficient and convenient approach for the preparation of EGBL enriched in SA but also accelerated efforts to high-value utilization of GBL.
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Ginkgo biloba , Extractos Vegetales , Hojas de la Planta , Ácido Shikímico , Ginkgo biloba/química , Hojas de la Planta/química , Extractos Vegetales/química , Ácido Shikímico/química , Adsorción , Cromatografía Líquida de Alta Presión/métodos , Porosidad , Flavonoides/análisis , Concentración de Iones de Hidrógeno , Extracto de GinkgoRESUMEN
Hyaluronic acid (HA), as a multifunctional hydrophilic polysaccharide, is potentially beneficial in improving the thermal stability of fermented modified starches, but relevant insights at the molecular level are lacking. The aim of this study was to investigate the effect of different levels (0 %, 3 %, 6 %, 9 %, 12 % and 15 %) of HA on the structural, thermal and pasting properties of wheat starch co-fermented with Saccharomyces cerevisiae and Lactobacillus plantarum. We found that the addition of HA increased the median particle size of fermented starch granules from 16.387 to 17.070 µm. Meanwhile, the crystallinity of fermented starch was negatively correlated with the HA content, decreasing from 14.70 % to 12.80 % (p < 0.05). Fourier transform infrared spectroscopy results confirmed that HA interacted with starch granules and water molecules mainly through hydrogen bonding. Thermal analyses showed that the thermal peak of the composite correlated with the HA concentration, reaching a maximum of 73.17 °C at 12 % HA. In addition, HA increases the pasting temperature, reduces the peak, breakdown and setback viscosities of starch. This study demonstrates the role of HA in improving the thermal stability of fermented starch, providing new insights for traditional fermented food research and the application of HA in food processing.
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Fermentación , Ácido Hialurónico , Lactobacillus plantarum , Saccharomyces cerevisiae , Almidón , Triticum , Lactobacillus plantarum/metabolismo , Saccharomyces cerevisiae/metabolismo , Almidón/química , Almidón/metabolismo , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Triticum/química , Temperatura , Espectroscopía Infrarroja por Transformada de Fourier , ViscosidadRESUMEN
The bromide-chloride mixed quasi-two-dimensional (2D) perovskite, with a natural quantum well structure and tunable exciton binding energy, has gained significant attention for high-performance blue perovskite light-emitting diodes (PeLEDs). However, the relative importance of having a low trap state density or efficient exciton transfer for high-efficiency electroluminescence (EL) performance remains elusive. Here, two molecules with the benzoic acid group, sodium 4-fluorobenzoate (SFB) and 3,5-dibromobenzoic acid (DBA), are used to modulate the phase distribution and trap state to explore the effect between energy transfer and defect passivation. As a result, when the n = 1 phase is inhibited in both films, the DBA@SFB-modified perovskite films achieve a higher photoluminescence quantum yield (PLQY) than the SFB-modified perovskite films due to effective defect passivation. However, DBA@SFB-modified PeLEDs exhibit lower external quantum efficiency (EQE) compared to SFB-modified PeLEDs due to the poor exciton transfer between the low-dimensional phase. This demonstrates that passivation strategies may enhance photoluminescence through reducing nonradiative recombination, but the effect of phase distribution is pivotal for EL performance by efficient energy transfer in quasi-2D perovskites. Femtosecond time-resolved transient absorption measurements confirm the fastest carrier dynamics in SFB-modified perovskite films, further corroborating the above result. This work provides useful information about phase modulation and defect passivation for high-efficiency blue quasi-2D PeLEDs.
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PURPOSE: Ultrasound is the imaging modality of choice for preoperative diagnosis of lymph node metastasis (LNM) in thyroid cancer (TC), yet its efficacy remains suboptimal. As radiomics gains traction in tumor diagnosis, its integration with ultrasound for LNM differentiation in TC has emerged, but its diagnostic merit is debated. This study assesses the accuracy of ultrasound-integrated radiomics in preoperatively diagnosing LNM in TC. METHODS: Literatures were searched in PubMed, Embase, Cochrane, and Web of Science until July 11, 2023. Quality of the studies was assessed by the radiomics quality score (RQS). A meta-analysis was executed using a bivariate mixed effects model, with a subgroup analysis based on modeling variables (clinical features, radiomics features, or their combination). RESULTS: Among 27 articles (16,410 TC patients, 6356 with LNM), the average RQS was 16.5 (SD:5.47). Sensitivity of the models based on clinical features, radiomics features, and radiomics features plus clinical features were 0.64, 0.76 and 0.69. Specificities were 0.77, 0.78 and 0.82. SROC values were 0.76, 0.84 and 0.81. CONCLUSION: Ultrasound-based radiomics effectively evaluates LNM in TC preoperatively. Adding clinical features does not notably enhance the model's performance. Some radiomics studies showed high bias, possibly due to the absence of standard application guidelines.
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Metástasis Linfática , Neoplasias de la Tiroides , Ultrasonografía , Humanos , Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/patología , Metástasis Linfática/diagnóstico por imagen , Ultrasonografía/métodos , Ganglios Linfáticos/diagnóstico por imagen , Ganglios Linfáticos/patología , Sensibilidad y Especificidad , RadiómicaRESUMEN
Nanoagents for chemodynamic therapy (CDT) hold a promising future in the field of antimicrobials, especially copper peroxide (CuO2) (CP) nanomaterials which have garnered significant attention due to their ability to self-supply H2O2. Nevertheless, the poor stability of CuO2 remains a critical challenge which restricts its practical application in the antibacterial field. In this study, an advanced nano-antimicrobial system HA-CP@Fe3O4 with enzyme-responsive properties is developed by coating hyaluronic acid (HA) on CuO2-loaded iron tetraoxide nanoparticles. The coating of HA not only stabilizes the CuO2 nanomaterials but also provides responsiveness towards the enzyme hyaluronidase, which is typically secreted by some bacteria. The outer layer of HA in HA-CP@Fe3O4 undergoes decomposition in the presence of hyaluronidase-secreting bacteria, resulting in the release of CuO2@Fe3O4. The released CuO2@Fe3O4 then self-supplies H2O2 and generates reactive oxygen species (ROS) within the infected microenvironment through Fenton and Russell effects, to ultimately achieve effective and precise antimicrobial activity. Simultaneously, the magnetic property provided by Fe3O4 allows the substance to be directed towards the infection site. Both in vitro and in vivo tests demonstrated that HA-CP@Fe3O4 exhibited excellent antimicrobial capabilities at low concentration (30 µg mL-1), exceptional biocompatibility and the ability to accelerate wound healing. The findings of this work offer a new and promising approach for targeted and precise CDT.
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Peróxido de Hidrógeno , Nanopartículas , Hialuronoglucosaminidasa , Antibacterianos/farmacología , Cicatrización de HeridasRESUMEN
We report on nonlinear terahertz third-harmonic generation (THG) measurements on YBa2Cu3O6+x thin films. Different from conventional superconductors, the THG signal starts to appear in the normal state, which is consistent with the crossover temperature T* of pseudogap over broad doping levels. Upon lowering the temperature, the THG signal shows an anomaly just below Tc in the optimally doped sample. Notably, we observe a beat pattern directly in the measured real-time waveform of the THG signal. We elaborate that the Higgs mode, which develops below Tc, couples to the mode already developed below T*, resulting in an energy level splitting. However, this coupling effect is not evident in underdoped samples. We explore different potential explanations for the observed phenomena. Our research offers valuable insight into the interplay between superconductivity and pseudogap.
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The addition of oat at varying percentages (26%, 32%, 38%, 44% and 50%) was used to evaluate the structural, microstructural, and physicochemical changes in instant-extruded rice (IER). A mixture of broken rice and oat flour was extruded in a twin-screw extruder. It was found that when adding 44% oats, the gelatinization degree of the mixed powder was the lowest (89.086 ± 1.966%). The dietary fiber content increased correspondingly with the increase in oat addition. Analyses of texture properties revealed that the hardness, adhesive, and resilience values increased and then decreased with oat addition. Compared with other common instant rice (IR), the advantages of IER were evaluated in terms of microstructure, digestive performance, and flavor. IER with 44% oat addition obtained in this study had higher hardness, adhesiveness, rehydration time, and sensory score, and the content of resistant starch (RS) reached 6.06%. The electronic nose and electronic tongue analyses could distinguish the flavor of different IR efficiently. This study showed the feasibility of preparing fiber-enriched IER. The results demonstrated the potential for the development and utilization of broken rice, providing a reference for the development of IER.
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d-Tetramethrin is one of the main components of mosquito control products, and is widely used for the control of dengue fever and insecticide production. Due to its widespread use, d-tetramethrin is a ubiquitous environmental pollutant and poses potential risks to human health. However, the effects of d-tetramethrin on liver morphology and function are not clearly established. In this study, we used zebrafish as an animal model to analyze the acute and chronic effects of d-tetramethrin exposure on the liver. We exposed zebrafish larvae and adults to different concentrations of d-tetramethrin and examined the impact of d-tetramethrin on lipid and glycogen metabolism, cellular properties, oxidative stress, cell proliferation, and apoptosis in the liver. We also analyzed transcriptional changes in genes related to apoptosis, inflammation, and cell proliferation using qPCR. Zebrafish exposed to d-tetramethrin exhibited severe liver damage, as evidenced by the presence of vacuoles and nuclear distortion in liver cells. The liver area in zebrafish larvae of the treatment group was significantly smaller than that of the control group. Significant lipid accumulation and decreased glycogen levels were observed in the livers of both zebrafish larvae and adults exposed to d-tetramethrin. Furthermore, d-tetramethrin exposure induced apoptosis and inflammation in zebrafish embryos. Additionally, d-tetramethrin caused liver damage, metabolic dysfunction, and impaired liver function. These results suggest that d-tetramethrin induces liver toxicity in zebrafish, by inducing oxidative stress and inhibiting cell proliferation.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Piretrinas , Pez Cebra , Animales , Humanos , Pez Cebra/metabolismo , Estrés Oxidativo , Inflamación , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Proliferación Celular , Glucógeno/metabolismo , Glucógeno/farmacología , Lípidos , LarvaRESUMEN
Nonlinear responses of superconductors to intense terahertz radiation has been an active research frontier. Using terahertz pump-terahertz probe spectroscopy, we investigate the c-axis nonlinear optical response of a high-temperature superconducting cuprate. After excitation by a single-cycle terahertz pump pulse, the reflectivity of the probe pulse oscillates as the pump-probe delay is varied. Interestingly, the oscillatory central frequency scales linearly with the probe frequency, a fact widely overlooked in pump-probe experiments. By theoretically solving the nonlinear optical reflection problem on the interface, we show that our observation is well explained by the Josephson-type third-order nonlinear electrodynamics, together with the emission coefficient from inside the material into free space. The latter results in a strong enhancement of the emitted signal whose physical frequency is around the Josephson plasma edge. Our result offers a benchmark for and new insights into strong-field terahertz spectroscopy of related quantum materials.
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In this paper, we use weak measurement to study the mixed magneto-optical Kerr effects (MOKEs) in a magnetic single layer of Co and in organic/Co bilayer films. The relationship between the amplified shift and the MOKE parameters is theoretically established and then experimentally observed as a function of both the magnetization intensity and the magnetization direction in magnetic thin films with an arbitrary magnetization. Furthermore, we experimentally observe a magnetic coupling at the organic/ferromagnetic interface. Due to this being a robust coupling, there is a significant dependence of the amplified shift with the thickness of the organic layer. This work has value for its application in the magnetic measurement technique for the more general case based on weak measurement.
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In order to find out the effect of salt concentration on fermented rape stalks, the physicochemical quality and volatile components was investigated using high performance liquid chromatography (HPLC) and headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results showed that there were abundant kinds of free amino acids (FAAs) in all samples, mainly presenting sweet, umami and bitter taste. Through taste activity value (TAV), His, Glu, and Ala contributed significantly to the taste of the sample. 51 volatile components were identified, of which the relative contents of ketones and alcohols were high. By the relative odor activity value (ROAV) analysis, the main components that had a great impact on the flavor were phenylacetaldehyde, ß-Ionone, ethyl palmitate and furanone. Adjusting the appropriate salt concentration for fermentation could improve the comprehensive quality of fermented rape stalks and promote the development and utilization of rape products.
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OBJECTIVE: This study aims to compare cognitive function and social functioning in male schizophrenia patients with deficit syndrome (DS) and non-DS, and to explore the associations among two different dimensions of negative symptoms (motivation and pleasure (MAP) and expressivity (EXP) deficits), cognitive function and social functioning base on a Structural Equation Model (SEM). METHODS: The current study enrolled 161 male schizophrenia patients and 120 age- and education- matched healthy controls. The DS and non-DS group were categorized by the Chinese version of Schedule for the Deficit Syndrome (SDS). The psychotic and negative symptoms were evaluated by the Brief Psychiatric Rating Scale (BPRS) and the Brief Negative Symptoms Scale (BNSS). The Social functioning was measured by Scale of Social function in Psychosis Inpatients (SSPI). A battery of classical neurocognitive tests was used for assessing cognition including sustained vigilance/attention, cognitive flexibility, ideation fluency and visuospatial memory. RESULTS: Our study indicated that DS patients performed worser in cognitive function and social functioning than non-DS patients. The SEM model demonstrated that MAP significantly affected social functioning through direct influence and mediation of cognitive function. However, our results found that EXP had little influence on cognitive function and social function. CONCLUSION: Our findings provided evidence supporting that DS may represent as a subtype of schizophrenia, and the MAP factor play a pivotal role to influence the cognitive and social functioning in schizophrenia patients.
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Esquizofrenia , Humanos , Masculino , Interacción Social , Motivación , Placer , Psicología del Esquizofrénico , Cognición , Pruebas Neuropsicológicas , Escalas de Valoración PsiquiátricaRESUMEN
Clear cell renal cell carcinoma (ccRCC) is the predominant type of kidney cancer, and the mutation of PBRM1 (Polybromo 1) gene is a commonly observed genetic alteration. The high frequency of PBRM1 mutation in ccRCC suggests its potential use as a biomarker for personalized therapy. In this study, we aimed to investigate the significance of PBRM1 mutation in disease progression and drug sensitivity in ccRCC. Additionally, we analyzed the critical pathways and genes associated with PBRM1 mutation to understand its potential mechanisms. Our findings show that PBRM1 mutation was observed in 38% of ccRCC patients and correlated with advanced disease stages. We also identified selective inhibitors for ccRCC with PBRM1 mutation using online databases such as PD173074 and AGI-6780. Furthermore, we identified 1253 genes as differentially expressed genes (DEGs) that were significantly enriched in categories such as metabolic progression, cell proliferation, and development. Although PBRM1 mutation did not show an association with ccRCC prognosis, a lower PBRM1 expression level correlated with worsened prognosis. Our study provides insights into the association of PBRM1 mutation with disease progression in ccRCC and suggests potential gene and signaling pathways for personalized treatment in ccRCC with PBRM1 mutation.
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We have studied the two phases of the molecular crystal acetylene, C2H2, using calculations of the lattice dynamics by Density Functional Theory methods. together with the use of classical molecular dynamics (MD) simulation methods. The two phases share the same simple face-centred cubic lattice arrangement of the molecular centres of mass, but with different molecular orientations. We show that the higher-temperature phase has lower phonon frequencies and hence higher entropy, giving thermodynamic stability at higher temperature. The calculated lattice dynamics of this phase show instabilities associated with phonons involving pure rotations of the molecules. The MD results show large amplitudes of librational motion in this phase. The MD simulations also showed a potential phase transition to a structure of tetragonal symmetry. The picture that emerges is that the phase transition in acetylene is a very rare example of one that encompasses elements of three types of transition: displacive, order-disorder and reconstructive.
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WDFY2 is a protein that may provide valuable insights into the mechanisms underlying human tumors and aid in the development of novel therapies. Despite its potential importance, the role of WDFY2 in pan-cancer has not been systematically investigated. In this study, we comprehensively explored the expression pattern and function of WDFY2 across 33 cancers using various databases, including TCGA, CPTAC and GEO datasets. Our results indicate that WDFY2 is downregulated in most cancer types, including BRCA, KIRP, KICH, LUAD, KIRC, PCPG, PRAD, THCA, ACC, OV, TGCT and UCS, while it is upregulated in CESC, CHOL, COAD, HNSC, LUSC, READ, STAD and UCEC. Prognostic analyses showed that higher levels of WDFY2 were associated with worse disease outcomes in ACC, BLCA, COAD, READ, SARC, MESO and OV. WDFY2 mutations were most frequent in colorectal cancer but were not associated with disease prognosis. We also found that WDFY2 expression correlated with monocyte infiltration status in SKCM, endothelial cell infiltration in COAD, KIRC, MESO, OV and THCA, and cancer-associated fibroblast infiltration in COAD, LUAD and OV. Additionally, functional enrichment analysis revealed that WDFY2 is involved in metabolism. Overall, our comprehensive analysis sheds light on the role of WDFY2 in various cancers, providing a better understanding of its role in tumorigenesis.
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To realize the precise modulation among the cesium lead halide perovskite-related phases is one of the most fascinating subjects and has motivated increasing research. The formation mechanisms of different phases of cesium lead halide have not been fully recognized. In this work, we reported the phase-selective synthesis of CsPbBr3 nanorods and Cs4PbBr6 nanocrystals (NCs) in amine-free systems, simply by adjusting the dosage of oleic acid (OA). By utilizing UV-visible absorption spectroscopy, we probed the evolution of the NCs and some lead bromide complex species during syntheses in reaction systems with different OA dosages. An OA-limited condition facilitated the formation of [PbBr4]2- and Cs4PbBr6 NCs. OA-rich conditions facilitated the formation of [PbBr3]-, [Pb2Br5]-, etc. at early stages and the subsequent generation of CsPbBr3 NCs. As the reaction systems equilibrated at a later stage, as-prepared CsPbBr3 or Cs4PbBr6 NCs would coexist with certain lead bromide complexes. OA dosage also greatly affected the kinetics of reactions toward CsPbBr3 NCs. A relatively large amount of OA would accelerate the formation of CsPbBr3 NCs. Our experimental results support two-step formation pathways of the cesium lead bromide NCs going through lead bromide complexes, and suggest that OA exerted all the influence by virtue of the lead bromide complexes. Our study presents a relatively clear picture of the formation of CsPbBr3 and Cs4PbBr6 NCs, which should be helpful in improving the preparation of lead halide perovskite-related NCs.
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Continuous emergence of persistent organic pollutants perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) in various water bodies around the world poses a serious threat to the global ecosystem. The exploration of advanced detection/removal techniques to monitor/treat such type of toxicants is urgently required. Herein, we unveiled a donor-acceptor type conjugated polymer PF-DBT-Im as a first-of-its-kind ratiometric fluorescent probe for visual, amplified, and specific monitoring of PFOA and PFOS with ultra-low detection limits of 6.12 nM (PFOA) and 14.3 nM (PFOS), respectively. PF-DBT-Im undergoes strong aggregation after binding with PFOA/PFOS as evident by transmission electron microscopy, zeta potential measurements, and dynamic light scattering studies. This promotes interchain Förster resonance energy transfer process to endorse an obvious emission color change from blue-to-magenta under ultraviolet lamp excitation. Consequently, a smartphone-integrated portable device is fabricated for realizing rapid and on-site detection of PFOA/PFOS. Besides, a new class of magnetic adsorbent Fe3O4@NH2&F13 is also prepared and used in combination with PF-DBT-Im to remove PFOA/PFOS from the environmental water effectively and rapidly as confirmed by liquid chromatography-mass spectrometry analysis. Thus, utilizing the excellent signal amplification property of PF-DBT-Im and the remarkable magnetic separation capability of Fe3O4@NH2&F13, a multifunctional system is developed for step-wise recognition and separation of PFOA/PFOS from the environmental water proficiently and rapidly.
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Ácidos Alcanesulfónicos , Fluorocarburos , Agua , Ecosistema , Ácidos Alcanesulfónicos/análisis , Fluorocarburos/análisis , Caprilatos/análisisRESUMEN
The advent of intense ultrashort optical pulses spanning a frequency range from terahertz to the visible has opened a new era in the experimental investigation and manipulation of quantum materials. The generation of strong optical field in an ultrashort time scale enables the steering of quantum materials nonadiabatically, inducing novel phenomenon or creating new phases which may not have an equilibrium counterpart. Ultrafast time-resolved optical techniques have provided rich information and played an important role in characterization of the nonequilibrium and nonlinear properties of solid systems. Here, some of the recent progress of ultrafast optical techniques and their applications to the detection and manipulation of physical properties in selected quantum materials are reviewed. Specifically, the new development in the detection of the Higgs mode and photoinduced nonequilibrium response in the study of superconductors by time-resolved terahertz spectroscopy are discussed.