RESUMEN
We have developed a DNA sensor that can be finalized to detect a specific target on demand. The electrode surface was modified with 2,7-diamino-1,8-naphthyridine (DANP), a small molecule with nanomolar affinity for the cytosine bulge structure. The electrode was immersed in a solution of synthetic probe-DNA that had a cytosine bulge structure at one end and a complementary sequence to the target DNA at the other end. The strong binding between the cytosine bulge and DANP anchored the probe DNAs to the electrode surface, and the electrode became ready for target DNA sensing. The complementary sequence portion of the probe DNA can be changed as requested, allowing for the detection of a wide variety of targets. Electrochemical impedance spectroscopy (EIS) with the modified electrode detected target DNAs with a high sensitivity. The charge transfer resistance (Rct) extracted from EIS showed a logarithmic relationship with the concentration of target DNA. The limit of detection (LoD) was less than 0.01 µM. By this method, highly sensitive DNA sensors for various target sequences could be easily produced.
Asunto(s)
Técnicas Biosensibles , Espectroscopía Dieléctrica , Ligandos , ADN/química , Sondas de ADN , Citosina , Electrodos , Técnicas Electroquímicas/métodosRESUMEN
Using the M13 phage display method, 236 amino acid sequences (peptide aptamers) that could specifically adsorb to CNTs were selected. These peptide aptamers had abundant hydrophobic amino acids and evenly dispersed charged amino acids. The hydrophobic amino acids were postulated to contribute to CNT adsorption, while the charged amino acids contribute to their aqueous solubility. The frequency of proline amino acids, which causes the amino acid main chain bending, was slightly higher than in nature, suggesting that some conformational constraint might be required. Four peptide aptamers with a high frequency of occurrence in the selected sequences were further studied. Hydrophobicity scores were periodic along the amino acid sequence. 3D structure predictions by PEP-FOLD3 indicated that these aptamers would take a helical structure with hydrophobic amino acid residues on one side, suggesting that the aptamers bind hydrophobically to the CNT. The adsorption of these four aptamers to the carbon electrode was confirmed by electrochemical impedance spectroscopy, which demonstrated the effectiveness of the phage display method. At the same time, it was shown that even for selected peptides, the adsorption performance varied, and verification was needed.
RESUMEN
We have found that the DNA intercalator [Ru(bpy)2DPPZ]2+ (bpy = 2,2'-bipyridine; DPPZ = dipyrido[3,2-a:2',3'-c]phenazine) causes an anomalous increase in charge transfer in electrochemical impedance spectroscopy (EIS). With a carbonaceous electrode and a 1 mM hexacyanoferrate (1 mM [Fe(CN)6]3- and 1 mM [Fe(CN)6]4-) mediator, we found that adding only 1 µM [Ru(bpy)2DPPZ]2+ greatly enhanced the charge transfer between the electrode and hexacyanoferrate mediator, independently of other electrolytes or buffer components. The effect started with a one millionth amount of hexacyanoferrate. Since [Ru(bpy)2DPPZ]2+ can intercalate with dsDNA, the effect is highly applicable for dsDNA detection or PCR monitoring. With further developments of this method, EIS sensors not requiring specific electrode modifications should be possible.
Asunto(s)
ADN , Sustancias Intercalantes , ADN/química , Electrodos , Sustancias Intercalantes/químicaRESUMEN
We developed a new electrochemical impedimetric method for the real-time detection of polymerase chain reactions (PCR) based on our recent discovery that the DNA intercalator, [Ru(bpy)2DPPZ]2+, anomalously enhances charge transfer between redox mediators, K4[Fe(CN)6]/K3[Fe(CN)6], and a carbon electrode. Three mM [Fe(CN)6]3-/4- and 5 µM [Ru(bpy)2DPPZ]2+ were added to the PCR solution, and electrochemical impedance spectroscopy (EIS) measurements were performed at each elongation heat cycle. The charge transfer resistance (Rct) was initially low due to the presence of [Ru(bpy)2DPPZ]2+ in the solution. As PCR progressed, amplicon dsDNA was produced exponentially, and intercalated [Ru(bpy)2DPPZ]2+ ions, which could be detected as a steep Rct, increased at specific heat cycles depending on the amount of template DNA. The Rct increase per heat cycle, ΔRct, showed a peak at the same heat cycle as optical detection, proving that PCR can be accurately monitored in real time by impedance measurement. This simple method will enable a cost-effective and portable PCR device.
Asunto(s)
Espectroscopía Dieléctrica , Sustancias Intercalantes , ADN/química , Espectroscopía Dieléctrica/métodos , Técnicas Electroquímicas , Sustancias Intercalantes/química , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
MicroRNAs (miRNAs) are short RNAs that regulate the expression of complementary messenger RNAs and are involved in numerous human diseases. However, current detection techniques lack the sensitivity to detect miRNAs of low abundance. Moreover, at a length of 20-25 bases, miRNAs are too short for the reverse transcription (RT) polymerase chain reaction (PCR). Here we have developed a new, rapid, and simple miRNA detection system utilizing an RT primer containing a DNA tag at the 5'-end to increase the length of the cDNA. This strategy increases the length of the hybridized tagged primer and the complementary template DNA, as well as the melting temperature of the primerâ template DNA duplex. PCR efficiency is thus increased, thereby enhancing miRNA detection sensitivity.
Asunto(s)
Cartilla de ADN/química , MicroARNs/análisis , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In this work, we demonstrate homogeneously distributed In0.3Ga0.7N/GaN quantum disks (QDs), with an average diameter below 10 nm and a high density of 2.1 × 10(11) cm(-2), embedded in 20 nm tall nanopillars. The scalable top-down fabrication process involves the use of self-assembled ferritin bio-templates as the etch mask, spin coated on top of a strained In0.3Ga0.7N/GaN single quantum well (SQW) structure, followed by a neutral beam etch (NBE) method. The small dimensions of the iron cores inside ferritin and nearly damage-free process enabled by the NBE jointly contribute to the observation of photoluminescence (PL) from strain-relaxed In0.3Ga0.7N/GaN QDs at 6 K. The large blueshift of the peak wavelength by over 70 nm manifests a strong reduction of the quantum-confined Stark effect (QCSE) within the QD structure, which also agrees well with the theoretical prediction using a 3D Schrödinger equation solver. The current results hence pave the way towards the realization of large-scale III-N quantum structures using the combination of bio-templates and NBE, which is vital for the development of next-generation lighting and communication devices.
RESUMEN
We produced a thermostable TiO2-(anatase)-coated multi-walled-carbon-nanotube (MWNT) nanocomposite for use in dye-sensitized solar cells (DSSCs) using biological supuramolecules as catalysts. We synthesized two different sizes of iron oxide nanoparticles (NPs) and arrayed the NPs on a silicon substrate utilizing two kinds of genetically modified cage-shaped proteins with silicon-binding peptide aptamers on their outer surfaces. Chemical vapor deposition (CVD) with the vapor-liquid-solid phase (VLS) method was applied to the substrate, and thermostable MWNTs with a diameter of 6 ± 1 nm were produced. Using a genetically modified cage-shaped protein with carbon-nanomaterials binding and Ti-mineralizing peptides as a catalyst, we were able to mineralize a titanium compound around the surface of the MWNT. The products were sintered, and thin TiO2-layer-coated MWNTs nanocomoposites were successfully produced. Addition of a 0.2 wt% TiO2-coated MWNT nanocomposite to a DSSC photoelectrode improved current density by 11% and decreased electric resistance by 20% compared to MWNT-free reference DSSCs. These results indicate that a nanoscale TiO2-layer-coated thermostable MWNT structure produced by our mutant proteins works as a superior electron transfer highway within TiO2 photoelectrodes.
Asunto(s)
Colorantes/química , Nanocompuestos/química , Nanotubos de Carbono/química , Fotoquímica/métodos , Titanio/química , Técnicas Electroquímicas , Electrones , Ferritinas/química , Ferritinas/genética , Nanotubos de Carbono/ultraestructura , Energía SolarRESUMEN
(Apo)ferritins are cage-shaped proteins which have recently received a great deal of attention because the inner cavity of the protein shell can be used as a size-restricted reaction field for the synthesis of nanomaterials. The biomineralization behavior and inorganic nanoparticle (NP) synthesis mechanism of (apo)ferritin in solution systems have been studied but the mineralization behavior of (apo)ferritin on the substrates has not yet been well studied. Here, we conducted quantitative and kinetic analyses of the mineralization behavior of immobilized (apo)ferritin on a polyelectrolyte multilayer (PEM) using quartz crystal microbalance (QCM), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS) techniques. We demonstrated that the (apo)ferritin immobilized on a substrate synthesizes a ferrihydrite core within the confines of the protein cage; similar to a solution dispersed system. In addition, we applied a ferritin/apoferritin blended monolayer to the study of iron mineralization and revealed that biomineralization in this system is spatially selective. It is important to understand the mineralization mechanisms for the synthesis of other functional NPs as this approach has potential for a broad range of magnetic, catalytic, and biomedical sensing applications.
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Ferritinas/química , Proteínas Inmovilizadas/química , Hierro/química , Minerales/química , Adsorción , Animales , Apoproteínas/química , Compuestos Férricos/química , Caballos , Microscopía Electrónica de Rastreo , Espectroscopía de Fotoelectrones , Tecnicas de Microbalanza del Cristal de CuarzoRESUMEN
The N-end rule pathway degrades proteins bearing a destabilization-inducing amino acid at the N-terminus. In this proteolytic system, Ubr ubiquitin ligases recognize and ubiquitylate substrates intended for degradation. Schizosaccharomyces pombe has two similar Ubr proteins, Ubr1 and Ubr11. Both proteins have unique roles in various cellular processes, although the ubr1∆ strain shows more severe defects. However, their involvement in the N-end rule pathway is unclear, and even the N-end rule pathway-dependent proteolytic activity has not been demonstrated in Sz. pombe. Here, we show that: (a) Sz. pombe has the N-end rule pathway in which only Ubr11, but not Ubr1, is responsible; and (b) the C-terminal fragment of the meiotic cohesin Rec8 (denoted as Rec8c) generated by separase-mediated cleavage is an endogenous substrate of the N-end rule pathway. Forced overexpression of stable Rec8c was deleterious in mitosis and caused a loss of the mini-chromosome. In unperturbed mitosis without overexpression, the rate of mini-chromosome loss was five-fold higher in the ubr11∆ strain. Since Rec8 is normally produced in meiosis, we examined whether meiosis and sporulation were affected in the ubr11∆ strain. In unperturbed meiosis, chromosome segregation occurred almost normally and viable spores were produced in the ubr11∆ cells, irrespective of the presence of undegraded endogenous Rec8c peptides.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Eliminación de Gen , Fosfoproteínas/metabolismo , Proteolisis , UbiquitinaciónRESUMEN
The interparticle distance between nanoparticles (NPs) dispersed on on SiO2 was shown to be controlled by PEGylation. Ferritins with nanoparticle cores were prepared and PEGylated with poly(ethylene glycol)s (PEGs) of two different molecular weights. It was shown that the thickness of the PEG layer on the ferritin surface determines the interparticle distance under short Debye lengths. Under conditions where the Debye length was greater than the PEG layer thickness, distance between ferritins increased due to the electrostatic repulsive force. Results suggest that the PEG layer accommodated a small amount of counterions insufficient to cancel the ferritin outer surface charges. Simulation showed that ferritins adsorbed randomly and interparticle distance can be predicted theoretically. We demonstrate that PEGylated ferritins, that is, NP cores, can be dispersed on a surface with interval distances between particles determined by the combination of the ionic strength of the solution and the molecular weight of the PEG.
Asunto(s)
Ferritinas/química , Ácido Poliglicólico/química , Adsorción , Peso Molecular , Tamaño de la Partícula , Dióxido de Silicio/química , Electricidad Estática , Propiedades de SuperficieRESUMEN
The first six peptides of multifunctional titanium binding peptide-1 bestowed recombinant L-ferritin, minT1-LF, was genetically engineered and used to fabricate multilayered nanoparticle architecture. The multifunctionality of minT1-LF enables specific binding of nanoparticle-accommodated minT1-LF to the silicon substrate surface and wet biochemical fabrication of gate oxide layer by its biomineralization activity. Three-dimensional (3D) nanoparticle architecture with multilayered structure was fabricated by the biological layer-by-layer method and embedded in a metal oxide-semiconductor device structure as a charge storage node of a flash memory device. The 3D-integrated multilayered nanoparticle architecture successfully worked as a charge storage node in flash memory devices that exhibited improved charge storage capacity compared with that of a conventional monolayer structure device.
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Nanoestructuras/química , Microscopía Electrónica de Transmisión , Nanoestructuras/ultraestructura , Semiconductores , VolatilizaciónRESUMEN
We successfully fabricated defect-free, distributed and sub-20-nm GaAs quantum dots (named GaAs nanodisks (NDs)) by using a novel top-down technique that combines a new bio-template (PEGylated ferritin) and defect-free neutral beam etching (NBE). Greater flexibility was achieved when engineering the quantum levels of ND structures resulted in greater flexibility than that for a conventional quantum dot structure because structures enabled independent control of thickness and diameter parameters. The ND height was controlled by adjusting the deposition thickness, while the ND diameter was controlled by adjusting the hydrogen-radical treatment conditions prior to NBE. Photoluminescence emission due to carrier recombination between the ground states of GaAs NDs was observed, which showed that the emission energy shift depended on the ND diameters. Quantum level engineering due to both diameter and thickness was verified from the good agreement between the PL emission energy and the calculated quantum confinement energy.
Asunto(s)
Arsenicales/química , Galio/química , Nanoestructuras/química , Nanotecnología/métodos , Puntos Cuánticos , Biotecnología/métodos , Ferritinas/química , Microscopía Electrónica de Rastreo , Nanoestructuras/ultraestructura , Tamaño de la Partícula , Polietilenglicoles/químicaRESUMEN
Gold nanoparticles are generally considered to be biologically inactive. However, in this study we show that the addition of 1.4 nm diameter gold nanoparticle induces the remodeling of the ring-shaped protein TRAP into a hollow, capsid-like configuration. This structural remodeling is dependent upon the presence of cysteine residues on the TRAP surface as well as the specific type of gold nanoparticle. The results reveal an apparent novel catalytic role of gold nanoparticles.
Asunto(s)
Cápside/química , Oro/química , Nanopartículas del Metal/química , Catálisis , Modelos Moleculares , Tamaño de la Partícula , Ingeniería de Proteínas , Propiedades de SuperficieRESUMEN
Cells adapt to oxidative stress by transcriptional activation of genes encoding antioxidants and proteins of other protective roles. A bZIP transcription factor, Pap1, plays a critical role in this process and overexpression of Pap1 confers resistance to various oxidants and drugs in fission yeast. Pap1 temporarily enters the nucleus upon oxidative stress but returns to the cytoplasm once cells adapt to the stress, suggesting that cellular localization regulates Pap1 function. We report here an additional regulatory mechanism that Ubr1 ubiquitin ligase-dependent degradation lowered the Pap1 protein levels. ubr1 cells were causally resistant to hydrogen peroxide because of the increment of Pap1 levels. Pap1 was preferentially degraded in the nucleus where Ubr1 was consistently enriched. Proteolysis was critical to downregulate Pap1 especially when its activation persisted, as constitutively nuclear Pap1 severely inhibited growth in ubr1 mutants. Inactive mutations in the bZIP DNA binding domain stabilized Pap1 but rescued the lethality caused by constitutively active Pap1 in ubr1 mutants. These findings indicate that either nuclear export or Ubr1-mediated proteolysis must be operative to prevent uncontrolled Pap1 function. Coincidental dysfunction in both inhibitory pathways causes lethality because of prolonged activation of Pap1. Ubr1 is a critical regulator for the homeostasis of oxidative stress response.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas Fúngicas/metabolismo , Estrés Oxidativo , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/fisiología , Estrés Fisiológico , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Núcleo Celular/química , Proteínas Fúngicas/genética , Técnicas de Inactivación de Genes , Peróxido de Hidrógeno/toxicidad , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Asociadas a PancreatitisRESUMEN
We demonstrate a chiral meta-molecule in the ultraviolet (UV) and visible (VIS) regions using a complex of Au nanoparticles (NPs) and rod-shaped tobacco mosaic virus (TMV). Au NPs five nm in diameter are uniformly formed on peptide-modified TMV. The peptide-modified TMV with uniform-sized Au NPs has improved dispersion in solution. A negative circular dichroism (CD) peak is produced around 540 nm, at plasmonic resonance wavelength of Au NPs. Additionally, modification of a CD peak in the UV region is observed. Attaching NPs to a virus causes the enhancement and modification of CD peaks in both the UV and VIS regions. Our results open a new avenue for the preparation of three dimensional chiral metamaterials at optical frequencies.
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Ingeniería Genética , Oro/química , Nanopartículas del Metal/química , Virus del Mosaico del Tabaco/química , Virus del Mosaico del Tabaco/genética , Absorción , Dicroismo Circular , Nanopartículas del Metal/ultraestructura , Soluciones , Espectrofotometría Ultravioleta , Virus del Mosaico del Tabaco/ultraestructuraRESUMEN
A sub-10 nm, high-density, periodic silicon-nanodisc (Si-ND) array has been fabricated using a new top-down process, which involves a 2D array bio-template etching mask made of Listeria-Dps with a 4.5 nm diameter iron oxide core and damage-free neutral-beam etching (Si-ND diameter: 6.4 nm). An Si-ND array with an SiO(2) matrix demonstrated more controllable optical bandgap energy due to the fine tunability of the Si-ND thickness and diameter. Unlike the case of shrinking Si-ND thickness, the case of shrinking Si-ND diameter simultaneously increased the optical absorption coefficient and the optical bandgap energy. The optical absorption coefficient became higher due to the decrease in the center-to-center distance of NDs to enhance wavefunction coupling. This means that our 6 nm diameter Si-ND structure can satisfy the strict requirements of optical bandgap energy control and high absorption coefficient for achieving realistic Si quantum dot solar cells.
RESUMEN
Biomineralization of ferritin core has been extended to the artificial synthesis of homogeneous metal complex nanoparticles (NPs) and semiconductor NPs. The inner cavity of apoferritin is an ideal spatially restricted chemical reaction chamber for NP synthesis. The obtained ferritin (biocomplexes, NP and the surrounding protein shell) has attracted great interest among researchers in the field of nanodevices. Ferritins were delivered onto specific substrate locations in a one-by-one manner or a hexagonally close-packed array through ferritin outer surface interactions. After selective elimination of protein shells from the ferritin, bare NPs were left at the positions where they were delivered. The obtained NPs were used as catalysts for carbon nanotube (CNT) growth and metal induced lateral crystallization (MILC), charge storage nodes of floating gate memory, and nanometer-scale etching masks, which could not be performed by other methods.
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Ferritinas/química , Ferritinas/fisiología , Nanoestructuras/química , Animales , Apoferritinas/química , Apoferritinas/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Estructura Molecular , Tamaño de la PartículaRESUMEN
We created a two-dimensional array of sub-10 nm Si-nanodiscs (Si-NDs), i.e. a 2D array of Si-NDs, with a highly ordered arrangement and dense NDs by using a new top-down technique comprising advanced damage-free neutral-beam (NB) etching and a bio-template (iron oxide core) as a uniform sub-10 nm etching mask. The bandgap energy (E(g)) of the fabricated 2D array of Si-NDs can be simply controlled from 2.2 to 1.3 eV by changing the ND thickness from 2 to 12 nm. Due to weak quantum confinement existing in the diameter direction resulting from the sub-10 nm Si-ND diameter, even though the thickness of the Si-ND is much larger than the Bohr radius of Si, E(g) is still larger than the 1.1 eV E(g) of bulk Si. Si-ND not only has wide controllable E(g) but also a high absorption coefficient due to quantum confinement in three dimensions. This new technique is a promising candidate for developing new nanostructures and could be integrated into the fabrication of nanoelectronic devices.
RESUMEN
This study reports controlled single conductive paths found in resistive random access memory (ReRAM) formed by embedding Pt nanoparticles (Pt NPs) in NiO film. Homogeneous Pt NPs produced and placed by ferritin protein produce electric field convergence which leads to controlled conductive path formation. The ReRAM with Pt NPs shows stable switching behavior. A Pt NP density decrease results in an increase of OFF state resistance and decrease of forming voltage, whereas ON resistance was independent of the Pt NP density, which indicates that a single metal NP in a memory cell will achieve low power and stable operation.
Asunto(s)
Ferritinas/química , Nanopartículas del Metal/química , Nanotecnología/métodos , Platino (Metal)/química , Impedancia Eléctrica , Nanopartículas del Metal/ultraestructuraRESUMEN
We used genetically modified tube-shaped tobamoviruses to produce 3 nm aligned magnetic nanoparticles. Amino acid residues facing the central channel of the virus were modified to increase the number of nucleation sites. Energy dispersive X-ray spectroscopy and superconducting quantum interference device analysis suggest that the particles consisted of Co-Pt alloy. The use of tobamovirus mutants is a promising approach to making a variety of components that can be applied to fabricate nanometer-scaled electronic devices.