RESUMEN
Researchers often consider microorganisms from Stenotrophomonas sp. to be beneficial for plants. In this study, the biocidal effects and action mechanisms of volatile organic compounds (VOCs) produced by Stenotrophomonas sp. NAU1697 were investigated. The mycelial growth and spore germination of Fusarium oxysporum f. sp. cucumerinum (FOC), which is a pathogen responsible for cucumber wilt disease, were significantly inhibited by VOCs emitted from NAU1697. Among the VOCs, 33 were identified, 11 of which were investigated for their antifungal properties. Among the tested compounds, 2-ethylhexanol exhibited the highest antifungal activity toward FOC, with a minimum inhibitory volume (MIV) of 3.0 µL/plate (equal to 35.7 mg/L). Damage to the hyphal cell wall and cell membrane integrity caused a decrease in the ergosterol content and a burst of reactive oxygen species (ROS) after 2-ethylhexanol treatment. DNA damage, which is indicative of apoptosis-like cell death, was monitored in 2-ethylhexanol-treated FOC cells by using micro-FTIR analysis. Furthermore, the activities of mitochondrial dehydrogenases and mitochondrial respiratory chain complex III in 2-ethylhexanol-treated FOC cells were significantly decreased. The transcription levels of genes associated with redox reactions and the cell wall integrity (CWI) pathway were significantly upregulated, thus indicating that stress was caused by 2-ethylhexanol. The findings of this research provide a new avenue for the sustainable management of soil-borne plant fungal diseases.
Asunto(s)
Fungicidas Industriales , Fusarium , Hexanoles , Enfermedades de las Plantas , Stenotrophomonas , Compuestos Orgánicos Volátiles , Fusarium/efectos de los fármacos , Fusarium/crecimiento & desarrollo , Compuestos Orgánicos Volátiles/farmacología , Compuestos Orgánicos Volátiles/química , Enfermedades de las Plantas/microbiología , Fungicidas Industriales/farmacología , Fungicidas Industriales/química , Hexanoles/farmacología , Hexanoles/química , Stenotrophomonas/efectos de los fármacos , Stenotrophomonas/genética , Stenotrophomonas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
4-Hexen-1-ol, 5-methyl-2-(1-methylethenyl)- was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, photoirritation/photoallergenicity, skin sensitization, and environmental safety. Data show that 4-hexen-1-ol, 5-methyl-2-(1-methylethenyl)- is not genotoxic. The repeated dose, reproductive, and local respiratory toxicity endpoints were evaluated using the Threshold of Toxicological Concern (TTC) for a Cramer Class I material, and the exposure to 4-hexen-1-ol, 5-methyl-2-(1-methylethenyl)- is below the TTC (0.03 mg/kg/day, 0.03 mg/kg/day, and 1.4 mg/day, respectively). Data from read-across analog 3-methylbut-3-en-1-ol (CAS # 763-32-6) show that there are no safety concerns for 4-hexen-1-ol, 5-methyl-2-(1-methylethenyl)- for skin sensitization under the current declared levels of use. The photoirritation/photoallergenicity endpoints were evaluated based on ultraviolet/visible (UV/Vis) spectra; 4-hexen-1-ol, 5-methyl-2-(1-methylethenyl)- is not expected to be photoirritating/photoallergenic. The environmental endpoints were evaluated; 4-hexen-1-ol, 5-methyl-2-(1-methylethenyl)- was found not to be Persistent, Bioaccumulative, and Toxic (PBT) as per the International Fragrance Association (IFRA) Environmental Standards, and its risk quotients, based on its current volume of use (VoU) in Europe and North America (i.e., Predicted Environmental Concentration/Predicted No Effect Concentration [PEC/PNEC]), are <1.
Asunto(s)
Perfumes , Animales , Humanos , Hexanoles/toxicidad , Hexanoles/química , Pruebas de Mutagenicidad , Odorantes , Perfumes/toxicidad , Perfumes/química , Medición de Riesgo , Pruebas de ToxicidadRESUMEN
An electrochemical aptamer-based sensor was developed for glutamate, the major excitatory neurotransmitter in the central nervous system. Determining glutamic acid release and glutamic acid levels is crucial for studying signal transmission and for diagnosing pathological conditions in the brain. Glutamic acid-selective oligonucleotides were isolated from an ssDNA library using the Capture-SELEX protocol in complex medium. The selection permitted the isolation of an aptamer 1d04 with a dissociation constant of 12 µM. The aptamer sequence was further used in the development of an electrochemical aptamer sensor. For this purpose, a truncated aptamer sequence named glu1 was labelled with a ferrocene redox tag at the 3'-end and immobilized on a gold electrode surface via Au-thiol bonds. Using 6-mercapto-1-hexanol as the backfill, the sensor performance was characterized by alternating current voltammetry. The glu1 aptasensor showed a limit of detection of 0.0013 pM, a wide detection range between 0.01 pM and 1 nM, and good selectivity for glutamate in tenfold diluted human serum. With this enzyme-free aptasensor, the highly selective and sensitive detection of glutamate was demonstrated, which possesses great potential for implementation in microelectrodes and for in vitro as well as in vivo monitoring of neurotransmitter release.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Electroquímicas/métodos , Ácido Glutámico/sangre , Técnicas Biosensibles/métodos , Ácido Glutámico/análisis , Hexanoles/química , Humanos , Límite de Detección , Compuestos de Sulfhidrilo/químicaRESUMEN
Tethered bilayer lipid membranes (tBLMs) have been known as stable and versatile experimental platforms for protein-membrane interaction studies. In this work, the assembly of functional tBLMs on silver substrates and the effect of the molecular chain-length of backfiller molecules on their properties were investigated. The following backfillers 3-mercapto-1-propanol (3M1P), 4-mercapto-1-butanol (4M1B), 6-mercapto-1-hexanol (6M1H), and 9-mercapto-1-nonanol (9M1N) mixed with the molecular anchor WC14 (20-tetradecyloxy-3,6,9,12,15,18,22 heptaoxahexatricontane-1-thiol) were used to form self-assembled monolayers (SAMs) on silver, which influenced a fusion of multilamellar vesicles and the formation of tBLMs. Spectroscopic analysis by SERS and RAIRS has shown that by using different-length backfiller molecules, it is possible to control WC14 anchor molecules orientation on the surface. An introduction of increasingly longer surface backfillers in the mixed SAM may be related to the increasing SAMs molecular order and more vertical orientation of WC14 at both the hydrophilic ethylenoxide segment and the hydrophobic lipid bilayer anchoring alkane chains. Since no clustering of WC14 alkane chains, which is deleterious for tBLM integrity, was observed on dry samples, the suitability of mixed-component SAMs for subsequent tBLM formation was further interrogated by electrochemical impedance spectroscopy (EIS). EIS showed the arrangement of well-insulating tBLMs if 3M1P was used as a backfiller. An increase in the length of the backfiller led to increased defectiveness of tBLMs. Despite variable defectiveness, all tBLMs responded to the pore-forming cholesterol-dependent cytolysin, vaginolysin in a manner consistent with the functional reconstitution of the toxin into phospholipid bilayer. This experiment demonstrates the biological relevance of tBLMs assembled on silver surfaces and indicates their utility as biosensing elements for the detection of pore-forming toxins in liquid samples.
Asunto(s)
Plata/química , Espectroscopía Dieléctrica , Hexanoles/química , Membrana Dobles de Lípidos/química , Espectrometría Raman , Compuestos de Sulfhidrilo/químicaRESUMEN
The modification of the biobased polymer chitosan is a broad and widely studied field. Herein, an insight into the hydrophobization of low-molecular-weight chitosan by substitution of amino functionalities with hexanoyl chloride is reported. Thereby, the influence of the pH of the reaction media was investigated. Further, methods for the determination of the degree of substitution based on 1H-NMR, FTIR, and potentiometric titration were compared and discussed regarding their accuracy and precision. 1H-NMR was the most accurate method, while FTIR and the potentiometric titration, though precise and reproducible, underlie the influence of complete protonation and solubility issues. Additionally, the impact of the pH variation during the synthesis on the properties of the samples was investigated by Cd2+ sorption experiments. The adjusted pH values during the synthesis and, therefore, the obtained degrees of substitution possessed a strong impact on the adsorption properties of the final material.
Asunto(s)
Quitosano/química , Acilación , Animales , Organismos Acuáticos , Hexanoles/química , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Layers formed from single-stranded DNA on nanostructured plasmonic metals can be applied as "working elements" in surface-enhanced Raman scattering (SERS) sensors used to sensitively and accurately identify specific DNA fragments in various biological samples (for example, in samples of blood). Therefore, the proper formation of the desired DNA layers on SERS substrates is of great practical importance, and many research groups are working to improve the process in forming such structures. In this work, we propose two modifications of a standard method used for depositing DNA with an attached linking thiol moiety on certain SERS-active structures; the modifications yield DNA layers that generate a stronger SERS signal. We propose: (i) freezing the sample when forming DNA layers on the nanoparticles, and (ii) when forming DNA layers on SERS-active macroscopic silver substrates, using ω-substituted alkanethiols with very short alkane chains (such as cysteamine or mercaptopropionic acid) to backfill the empty spaces on the metal surface unoccupied by DNA. When 6-mercapto-1-hexanol is used to fill the unoccupied places on a silver surface (as in experiments on standard gold substrates), a quick detachment of chemisorbed DNA from the silver surface is observed. Whereas, using ω-substituted alkanethiols with a shorter alkane chain makes it possible to easily form mixed DNA/backfilling thiol monolayers. Probably, the significantly lower desorption rate of the thiolated DNA induced by alkanethiols with shorter chains is due to the lower stabilization energy in monolayers formed from such compounds.
Asunto(s)
ADN de Cadena Simple/química , Oro/química , Plata/química , Hexanoles/química , Nanopartículas del Metal/química , Espectrometría Raman , Compuestos de Sulfhidrilo/química , Propiedades de SuperficieRESUMEN
The present study investigates volatile organic compound (VOC) compositional changes in Liuyang Douchi during fermentation via a HS-GC-IMS and HS-SPME-GC-MS combination approach. A total of 115 VOCs were identified from Douchi, most of which were accumulated during pile fermentation. Notably, most alcohols and acids decreased with fermentation, while esters, ketones, pyrazines, and phenols accumulated during pile fermentation. Depending on the VOCs identified by GC-IMS/MS, the different fermentation stages of Douchi could be facilely distinguished. Of these, 49 VOCs were regarded as the marker VOCs of Douchi in different fermentation stage: hexanol, hexanal, and propanoic acid was the marker VOCs of the black beans before fermentation and contributing beany and grassy odors; 1-octen-3-ol and 3-octanone supplying a mushroom aroma to the Douchi fermented for 3-9 days; and esters and pyrazine, especially ethyl acetate and 2,6-dimethylpyrazine, contributing the cocoa, fruity, and nutty aromas of matured Douchi.
Asunto(s)
Alimentos Fermentados/análisis , Glycine max/química , Alimentos de Soja/análisis , Compuestos Orgánicos Volátiles/química , Aldehídos/química , Fermentación , Cromatografía de Gases y Espectrometría de Masas , Hexanoles/química , Odorantes , Propionatos/química , Microextracción en Fase Sólida , GustoRESUMEN
Aphidius gifuensis Ashmaed is a generalist endoparasitoid that parasitizes a variety of aphid species. In China, it is widely used as a biological control agent to protect vegetables and tobaccos in open fields; control efficiency is largely dependent on its host-seeking ability. In this study, a six-choice olfactometer was used to investigate the olfactory responses of A. gifuensis to tobacco plants that had suffered damage (either varying degrees of mechanical damage or from aphid-feeding at different time intervals) and tobacco volatiles with different dosages. Furthermore, the regularity of A. gifuensis females' response toward an aphid/tobacco complex was monitored using a Y-tube olfactometer. Our findings suggest that tobacco plants are significantly attractive to A. gifuensis after they have been punctured with 50 holes, or housed with Myzus persicae (Sulzer) at a density of 400 aphids, except at an infestation time of 12 h. Moreover, aphid density had a more significant effect on the response than the time interval since aphid application. Aphidius gifuensis was found to be active during the daytime and preferred to search for their aphid hosts at 14:00 h. Five EAG-active tobacco volatiles (trans-2-hexenal, methyl salicylate, benzaldehyde, cis-3-hexen-1-ol, and 1-hexanal) were found to significantly attract A. gifuensis females at different concentration ranges. The practical implications of these results are discussed in the framework of the sustainable biological control of pest aphids in agricultural production systems.
Asunto(s)
Áfidos , Señales (Psicología) , Conducta de Búsqueda de Hospedador/fisiología , Compuestos Orgánicos Volátiles , Avispas/fisiología , Animales , Áfidos/metabolismo , Áfidos/parasitología , Agentes de Control Biológico , China , Productos Agrícolas , Hexanoles/química , Hexanoles/metabolismo , Olfatometría , Parásitos/fisiología , Control Biológico de Vectores , Olfato , Nicotiana/metabolismo , Compuestos Orgánicos Volátiles/química , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
Ambrosia beetles (Coleoptera: Scolytinae) cultivate their fungal symbiont within host substrates as the sole source of nutrition on which the larvae and adults must feed. To investigate a possible role for semiochemicals in this interaction, we characterized electrophysiological and behavioral responses of Xylosandrus germanus to volatiles associated with its fungal symbiont Ambrosiella grosmanniae. During still-air walking bioassays, X. germanus exhibited an arrestment response to volatiles of A. grosmanniae, but not antagonistic fungi Beauveria bassiana, Metarhizium brunneum, Trichoderma harzianum, the plant pathogen Fusarium proliferatum, or malt extract agar. Solid phase microextraction-gas chromatography-mass spectrometry identified 2-ethyl-1-hexanol, 2-phenylethanol, methyl benzoate and 3-methyl-1-butanol in emissions from A. grosmanniae; the latter two compounds were also detected in emissions from B. bassiana. Concentration-responses using electroantennography documented weak depolarizations to A. grosmanniae fungal volatiles, unlike the comparatively strong response to ethanol. When tested singly in walking bioassays, volatiles identified from A. grosmanniae elicited relatively weak arrestment responses, unlike the responses to ethanol. Xylosandrus germanus also exhibited weak or no long-range attraction to the fungal volatiles when tested singly during field trials in 2016-2018. None of the fungal volatiles enhanced attraction of X. germanus to ethanol when tested singly; in contrast, 2-phenylethanol and 3-methyl-1-butanol consistently reduced attraction to ethanol. Volatiles emitted by A. grosmanniae may represent short-range olfactory cues that could aid in distinguishing their nutritional fungal symbiont from other fungi, but these compounds are not likely to be useful as long-range attractants for improving detection or mass trapping tactics.
Asunto(s)
Feromonas/química , Compuestos Orgánicos Volátiles/química , Animales , Ascomicetos/metabolismo , Conducta Animal , Benzoatos/química , Benzoatos/metabolismo , Evolución Biológica , Fenómenos Electrofisiológicos , Etanol/química , Etanol/metabolismo , Femenino , Fusarium/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Hexanoles/química , Hexanoles/metabolismo , Control de Insectos , Pentanoles/química , Pentanoles/metabolismo , Feromonas/metabolismo , Microextracción en Fase Sólida , Simbiosis , Compuestos Orgánicos Volátiles/metabolismo , GorgojosRESUMEN
Two new hexenol glycosides, (Z)-hex-3-en-1-ol O-ß-d-xylcopyranosyl-(1-6)-ß -d-glucopyranosyl-(1-2)-ß-d-glucopyranoside (1) and (E)-hex-3-en-1-ol O-ß-d-xylcopyranosyl-(1-6)-ß-d-glucopyranosyl-(1-2)-ß-d-glucopyranoside (2), were isolated from the 50% ethanol elution of macroporous resin of Physalis alkekengi var. franchetii. Their structures were established by detailed spectroscopic analysis, including extensive 2D-NMR data. This is the first time to report the (Z) and (E) 3-hexenol glycosides from Physalis alkekengi var. franchetii.
Asunto(s)
Antibacterianos/farmacología , Glicósidos/química , Glicósidos/farmacología , Physalis/química , Antibacterianos/química , Evaluación Preclínica de Medicamentos , Flores/química , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Hexanoles/química , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Resinas de Plantas/química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The aroma changes in instant white tea resulting from ß-glucosidase treatment was investigated by quantitative descriptive analysis (QDA), gas chromatography-mass spectrometry (GC-MS), odour activity value analysis (OAV), aroma reconstruction and omission tests. The grassy, floral and sweet notes increased significantly (P < 0.05), and the roasted note decreased significantly (P < 0.05) upon ß-glucosidase treatment. Quantitative analysis showed that the concentrations of benzaldehyde, benzeneacetaldehyde, (Z)-3-hexen-1-ol, linalool, phenylethyl alcohol, cis-linalool oxide, trans-linalool oxide, hexanol, hotrienol and (E)-2-hexen-1-ol increased significantly (P < 0.05) after treatment; however, (Z)-3-hexen-1-ol isomerized to (E)-2-hexen-1-ol. OAV analysis, aroma reconstruction and the omission test showed that the grassy, floral and sweet notes increased as the (Z)-3-hexen-1-ol, cis/trans-linalool oxide and benzeneacetaldehyde increased, whereas the roasted note declined under the same conditions. The enzymatic hydrolysis of glycosidic precursors and the auto-isomerization of volatile compounds provide new information for understanding how ß-glucosidase treatment improves the aroma of tea products.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas , Té/metabolismo , Compuestos Orgánicos Volátiles/química , beta-Glucosidasa/metabolismo , Hexanoles/química , Hidrólisis , Isomerismo , Odorantes/análisis , Té/química , Termodinámica , Compuestos Orgánicos Volátiles/análisisRESUMEN
Recent years have seen the development of a number of biosensor architectures that rely on target binding-induced changes in the rate of electron transfer from an electrode-bound receptor. Most often, the interrogation of these sensors has relied on voltammetric methods, such as square-wave voltammetry, which limit their time resolution to a few seconds. Here, we describe the use of an impedance-based approach, which we have termed electrochemical phase interrogation, as a means of collecting high time resolution measurements with sensors in this class. Specifically, using changes in the electrochemical phase to monitor target binding in an electrochemical-aptamer based (EAB) sensor, we achieve subsecond temporal resolution and multihour stability in measurements performed directly in undiluted whole blood. Electrochemical phase interrogation also offers improved insights into EAB sensors' signaling mechanism. By modeling the interfacial resistance and capacitance using equivalent circuits, we find that the only parameter that is altered by target binding is the charge-transfer resistance. This confirms previous claims that binding-induced changes in electron-transfer kinetics drive signaling in this class of sensors. Considering that a wide range of electrochemical biosensor architectures rely on this signaling mechanism, we believe that electrochemical phase interrogation may prove generalizable toward subsecond measurements of molecular targets.
Asunto(s)
Aptámeros de Nucleótidos/química , Tobramicina/sangre , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Técnicas Electroquímicas , Hexanoles/química , Cinética , Modelos Químicos , Conformación Molecular , Oxidación-Reducción , Compuestos de Sulfhidrilo/química , Propiedades de SuperficieRESUMEN
The 2-ethylhexyl stearate is used as a bio-lubricant in various cosmetic products. The present study is focused on the biocatalyzed esterification of 2-ethylhexanol and stearic acid to form 2-ethylhexyl stearate catalyzed by Fermase CALB 10000 in the presence of ultrasound treatment. The maximum conversion (95.87%) was obtained at molar ratio of 2-ethylhexanol to stearic acid 2:1, enzyme amount of 2 % (w/w), power 80 W, duty cycle 50 % and temperature 50°C in comparatively short reaction time (3 h) in the presence of Fermase as a catalyst. At optimum conditions, it is observed that in the presence of ultrasound; the reaction time minimizes up to 4 h as compared to mechanical stirring method (7 h). The physiochemical properties for the 2-ethylhexyl palmitate were also evaluated.
Asunto(s)
Hexanoles/química , Estearatos/síntesis química , Ondas Ultrasónicas , Biocatálisis , Fenómenos Químicos , Enzimas Inmovilizadas/química , Esterificación , Estearatos/química , Ácidos Esteáricos/química , Temperatura , Factores de TiempoRESUMEN
Reconstituted polyphenolic and aromatic fractions (PAFs) from 33 different Garnacha and Tempranillo grapes were incubated in strict anoxia (75 °C × 24 h). Obtained hydrolyzates were characterized by sensory analysis, gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS). Five different aroma categories emerged. Garnacha may develop specific tropical/citrus fruit, kerosene and floral and Tempranillo toasty-woody and red-fruit characteristics. Those notes seem to mask alcoholic and fruit-in-syrup descriptors and the common vegetal background. Twenty-seven odorants were detected by GC-O. GC-MS data showed a clustering closely matching the one found by sensory analysis, suggesting the existence of five specific metabolomic profiles behind the five specific sensory profiles. Overall results suggest that 3-mercaptohexanol is responsible for tropical/citrus fruit, TDN for kerosene, volatile phenols for woody/toasty, ß-damascenone and massoia lactone, likely with Z-1,5-octadien-3-one for fruit-in-syrup and alcoholic notes. Nine lipid-derived unsaturated aldehydes and ketones may be responsible for the vegetal background.
Asunto(s)
Vitis/química , Compuestos Orgánicos Volátiles/análisis , Frutas/química , Cromatografía de Gases y Espectrometría de Masas/métodos , Hexanoles/química , Norisoprenoides/química , Odorantes/análisis , Olfatometría , Análisis de Componente Principal , Compuestos de Sulfhidrilo/químicaRESUMEN
The chemical composition of the volatile fraction from Galium verum L. (leaves and flowers) and Cruciata laevipes Opiz (whole plant), Rubiaceae, was investigated. Samples from these two plant species were collected at full bloom in Val di Susa (Western Alps, Turin, Italy), distilled in a Clevenger-type apparatus, and analyzed by GC/FID and GC/MS. A total of more than 70 compounds were identified, making up 92%-98% of the total oil. Chemical investigation of their essential oils indicated a quite different composition between G. verum and C. laevipes, both in terms of the major constituents and the dominant chemical classes of the specialized metabolites. The most abundant compounds identified in the essential oils from G. verum were 2-methylbenzaldheyde (26.27%, corresponding to 11.59 µg/g of fresh plant material) in the leaves and germacrene D (27.70%; 61.63 µg/g) in the flowers. C. laevipes essential oils were instead characterized by two sesquiterpenes, namely ß-caryophyllene (19.90%; 15.68 µg/g) and trans-muurola-4(15),5-diene (7.60%; 5.99 µg/g); two phenylpropanoids, benzyl alcohol (8.30%; 6.71 µg/g), and phenylacetaldehyde (7.74%; 6.26 µg/g); and the green-leaf alcohol cis-3-hexen-1-ol (9.69%; 7.84 µg/g). The ecological significance of the presence of such compounds is discussed.
Asunto(s)
Flores/química , Galium/química , Hojas de la Planta/química , Rubiaceae/química , Compuestos Orgánicos Volátiles/aislamiento & purificación , Acetaldehído/análogos & derivados , Acetaldehído/química , Acetaldehído/aislamiento & purificación , Altitud , Benzaldehídos/química , Benzaldehídos/aislamiento & purificación , Alcohol Bencilo/química , Alcohol Bencilo/aislamiento & purificación , Cromatografía de Gases y Espectrometría de Masas , Hexanoles/química , Hexanoles/aislamiento & purificación , Humanos , Italia , Extracción Líquido-Líquido/métodos , Aceites de Plantas/química , Sesquiterpenos Policíclicos/química , Sesquiterpenos Policíclicos/aislamiento & purificación , Sesquiterpenos de Germacrano/química , Sesquiterpenos de Germacrano/aislamiento & purificación , Compuestos Orgánicos Volátiles/química , Compuestos Orgánicos Volátiles/clasificaciónRESUMEN
Recent work has gradually been clarifying the binding site of non-electrophilic agonists on the transient receptor potential A1 (TRPA1). This study searched for non-electrophilic TRPA1 agonists by means of in silico drug discovery techniques based on three-dimensional (3-D) protein structure. First, agonist-bound pocket structures were explored using an advanced molecular dynamics simulation starting from the cryo-electron microscopic structure of TRPA1, and several pocket structures suitable for virtual screening were extracted by structure evaluation using known non-electrophilic TRPA1 agonists. Next, 49 compounds were selected as new non-electrophilic agonist candidates from a library of natural products comprising 10,555 compounds by molecular docking toward these pocket structures. Measurement of the TRPA1 agonist activity of these compounds showed notable TRPA1 activation with three compounds (decanol, 2-ethyl-1-hexanol, phenethyl butanoate). Decanol and 2-ethyl-1-hexanol, which are categorized as fatty alcohols, in particular have a novel chemical scaffold for TRPA1 activation. The results of this study are expected to be of considerable use in understanding the molecular mechanism of TRPA1 recognition by non-electrophilic agonists.
Asunto(s)
Productos Biológicos/química , Canal Catiónico TRPA1/agonistas , Sitios de Unión , Productos Biológicos/metabolismo , Hexanoles/química , Hexanoles/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Canal Catiónico TRPA1/metabolismoRESUMEN
Electrochemical aptamer-based (E-AB) sensors achieve highly precise measurements of specific molecular targets in untreated biological fluids. This unique ability, together with their measurement frequency of seconds or faster, has enabled the real-time monitoring of drug pharmacokinetics in live animals with unprecedented temporal resolution. However, one important weakness of E-AB sensors is that their bioelectronic interface degrades upon continuous electrochemical interrogation-a process typically seen as a drop in faradaic and an increase in charging currents over time. This progressive degradation limits their in vivo operational life to 12 h at best, a period that is much shorter than the elimination half-life of the vast majority of drugs in humans. Thus, there is a critical need to develop novel E-AB interfaces that resist continuous electrochemical interrogation in biological fluids for prolonged periods. In response, our group is pursuing the development of better packed, more stable self-assembled monolayers (SAMs) to improve the signaling and extend the operational life of in vivo E-AB sensors from hours to days. By invoking hydrophobicity arguments, we have created SAMs that do not desorb from the electrode surface in aqueous physiological solutions and biological fluids. These SAMs, formed from 1-hexanethiol solutions, decrease the voltammetric charging currents of E-AB sensors by 3-fold relative to standard monolayers of 6-mercapto-1-hexanol, increase the total faradaic current, and alter the electron transfer kinetics of the platform. Moreover, the stability of our new SAMs enables uninterrupted, continuous E-AB interrogation for several days in biological fluids, like undiluted serum, at a physiological temperature of 37 °C.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Compuestos de Sulfhidrilo/química , Técnicas Biosensibles/instrumentación , Técnicas Electroquímicas/instrumentación , Electrodos , Hexanoles/química , Humanos , Suero/químicaRESUMEN
To harness the applicability of microribonucleic acid (miRNA) as a cancer biomarker, the detection sensitivity of serum miRNA needs to be improved. This study evaluated the detection sensitivity of miRNA hybridization using cyclic voltammograms (CVs) and microelectrode array chips modified with peptide nucleic acid (PNA) probes and 6-hydroxy-1-hexanethiol. We investigated the PNA probe modification pattern on array chips using fluorescently labeled cDNA. The pattern was not uniformly spread over the working electrode (WE) and had a one-dimensional swirl-like pattern. Accordingly, we established a new ion-channel sensor model wherein the WE is negatively biased through the conductive π-π stacks of the PNA/DNA duplexes. This paper discusses the mechanism underlying the voltage shift in the CV curves based on the electric double-layer capacitance. Additionally, the novel hybridization evaluation parameter ΔE is introduced. Compared to conventional evaluation using oxidation current changes, ΔE was more sensitive. Using ΔE and a new hybridization system for ultrasmall amounts of aqueous solutions (as low as 35 pL), 140 zeptomol label-free miRNA were detected without polymerase chain reaction (PCR) amplification at an adequate sensitivity. Herein, the differences in the target molar amount and molar concentration are elucidated from the viewpoint of hybridization sensitivity.
Asunto(s)
Capacidad Eléctrica , Electroquímica/métodos , MicroARNs/sangre , Hibridación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/química , Técnicas Biosensibles/métodos , Conductividad Eléctrica , Electrodos , Hexanoles/química , Humanos , Iones , Microelectrodos , Microscopía Electrónica de Rastreo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Compuestos de Sulfhidrilo/químicaRESUMEN
Despite a large number of publications describing biosensors based on electrochemical impedance spectroscopy (EIS), little attention has been paid to the stability and reproducibility issues of the sensor interfaces. In this work, the stability and reproducibility of faradaic EIS analyses on the aptamer/mercaptohexanol (MCH) self-assembled monolayer (SAM)-functionalized gold surfaces in ferri- and ferrocyanide solution were systematically evaluated prior to and after the aptamer-probe DNA hybridization. It is shown that the EIS data exhibited significant drift, and this significantly affected the reproducibility of the EIS signal of the hybridization. As a result, no significant difference between the charge transfer resistance (RCT) changes induced by the aptamer-target DNA hybridization and that caused by the drift could be identified. A conditioning of the electrode in the measurement solution for more than 12 h was required to reach a stable RCT baseline prior to the aptamer-probe DNA hybridization. The monitored drift in RCT and double layer capacitance during the conditioning suggests that the MCH SAM on the gold surface reorganized to a thinner but more closely packed layer. We also observed that the hot binding buffer used in the following aptamer-probe DNA hybridization process could induce additional MCH and aptamer reorganization, and thus further drift in RCT. As a result, the RCT change caused by the aptamer-probe DNA hybridization was less than that caused by the hot binding buffer (blank control experiment). Therefore, it is suggested that the use of high temperature in the EIS measurement should be carefully evaluated or avoided. This work provides practical guidelines for the EIS measurements. Moreover, because SAM-functionalized gold electrodes are widely used in biosensors, for example, DNA sensors, an improved understanding of the origin of the observed drift is very important for the development of well-functioning and reproducible biosensors.