RESUMO
Quantum dots (QDs) are considered for devices like light-emitting diodes (LEDs) and photodetectors as a result of their tunable optoelectronic properties. To utilize the full potential of QDs for optoelectronic applications, control over the charge carrier density is vital. However, controlled electronic doping of these materials has remained a long-standing challenge, thus slowing their integration into optoelectronic devices. Electrochemical doping offers a way to precisely and controllably tune the charge carrier concentration as a function of applied potential and thus the doping levels in QDs. However, the injected charges are typically not stable after disconnecting the external voltage source because of electrochemical side reactions with impurities or with the surfaces of the QDs. Here, we use photopolymerization to covalently bind polymerizable electrolyte ions to polymerizable solvent molecules after electrochemical charge injection. We discuss the importance of using polymerizable dopant ions as compared to nonpolymerizable conventional electrolyte ions such as LiClO4 when used in electrochemical doping. The results show that the stability of charge carriers in QD films can be enhanced by many orders of magnitude, from minutes to several weeks, after photochemical ion fixation. We anticipate that this novel way of stable doping of QDs will pave the way for new opportunities and potential uses in future QD electronic devices.
RESUMO
Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.
Assuntos
Análise de Sequência de Proteína/métodos , Imagem Individual de Molécula/métodos , Espectrometria de Massas/métodos , Nanotecnologia , Proteínas/química , Proteômica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodosRESUMO
While DNA sequencing is now amply available, fast, and inexpensive, protein sequencing remains a tremendous challenge. Nanopores may allow for developing a protein sequencer with single-molecule capabilities. As identification of 20 different amino acids currently presents an unsurmountable challenge, fingerprinting schemes are pursued, in which only a subset of amino acids is labeled and detected. This requires modification of amino acids with chemical structures that generate a distinct nanopore ionic current signal. Here, we use a model peptide and the fragaceatoxin C nanopore to characterize six potential tags for a fingerprinting approach using nanopores. We find that labeled and unlabeled proteins can be clearly distinguished and that sensitive detection is obtained for labels with a spectrum of different physicochemical properties such as mass (427-1275 Da), geometry, charge, and hydrophobicity. Additionally, information about the position of the label along the peptide chain can be obtained from individual current-blockade event features. The results represent an important advance toward the development of a single-molecule protein-fingerprinting device with nanopores.
Assuntos
Aminoácidos/química , Venenos de Cnidários/química , Nanoporos , Peptídeos/química , Sequência de Aminoácidos , Fluoresceína/químicaRESUMO
Two molecular beacons were designed as complementary fluorescent imaging probes for miRNA-21 and miRNA-31. Both beacons were prepared by a combination of solid-phase protocol and Cu(i)-catalyzed cycloaddition chemistry. The four photostable and bright fluorophores were attached to 2'-positions in the stem part of the two beacons. One beacon was labeled by a green-to-red emitting and the other by a blue-to-yellow emitting energy transfer pair. This two by two combination yields the four color emission readout. In vitro experiments demonstrate rapid and highly selective opening of both molecular beacons upon addition of the complementary target RNA and excellent green : red and blue : yellow emission color contrasts. Confocal microscopy of selected cancer cell lines provides evidence that a four color imaging of versicular miRNA-21 and miRNA-31 can be achieved both selectively and simultaneously upon transfection by the beacons, and that the fluorescent readouts track well with miRNA levels determined by PCR.
Assuntos
MicroRNAs/metabolismo , Sondas de Oligonucleotídeos/metabolismo , Imagem Óptica/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Espaço Intracelular/metabolismo , Sondas de Oligonucleotídeos/química , Fatores de TempoRESUMO
The so-called acpcPNA system bears a peptide backbone consisting of 4'-substituted proline units with (2'R,4'R) configuration in an alternating combination with (2S)-amino-cyclopentane-(1S)-carboxylic acids. acpcPNA forms exceptionally stable hybrids with complementary DNA. We demonstrate herein (i) strand displacements by single-stranded DNA from acpcPNA-DNA hybrids, and by acpcPNA strands from DNA duplexes, and (ii) strand invasions by acpcPNA into double-stranded DNA. These processes were studied in vitro using synthetic oligonucleotides and by means of our concept of wavelength-shifting fluorescent nucleic acid probes, including fluorescence lifetime measurements that allow quantifying energy transfer efficiencies. The strand displacements of preannealed 14mer acpcPNA-7mer DNA hybrids consecutively by 10mer and 14mer DNA strands occur with rather slow kinetics but yield high fluorescence color ratios (blue : yellow or blue : red), fluorescence intensity enhancements, and energy transfer efficiencies. Furthermore, 14mer acpcPNA strands are able to invade into 30mer double-stranded DNA, remarkably with quantitative efficiency in all studied cases. These processes can also be quantified by means of fluorescence. This remarkable behavior corroborates the extraordinary versatile properties of acpcPNA. In contrast to conventional PNA systems which require 3 or more equivalents PNA, only 1.5 equivalents acpcPNA are sufficient to get efficient double duplex invasion. Invasions also take place even in the presence of 250 mM NaCl which represents an ionic strength nearly twice as high as the physiological ion concentration. These remarkable results corroborate the extraordinary properties of acpcPNA, and thus acpcPNA represents an eligible tool for biological analytics and antigene applications.
Assuntos
DNA/química , Ácidos Nucleicos Peptídicos/química , Sequência de Bases , DNA/genética , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/genéticaRESUMO
DNA-based aptamers are commonly used recognition elements in biosensors for a range of target molecules. Here, the development of a wavelength-shifting optical module for a DNA-based adenosine-binding aptamer is described. It applies the combination of two photostable cyanine-styryl dyes as covalent modifications. This energy-transfer pair is postsynthetically attached to oligonucleotides via a copper(I)-catalyzed azide-alkyne cycloaddition by two structurally different approaches: 1)â as nucleotide modifications at the 2'-position of uridines and 2)â as nucleotide substitutions using (S)-amino-1,2-propanediol as acyclic linker between the phosphodiester bridges. Both dyes exhibit a remarkable photostability. A library of DNA aptamers consisting of different combinations of the two dyes in diagonal orientations were evaluated by their emission color contrast as readout. Further optimization led to aptasensors with improved fluorescent readout as compared with previously reported aptasensors. This approach described is synthetically facile using simple propargylated phosphoramidites as DNA building blocks. As such, this approach could be applied for other dyes and other chemical/biological applications.
RESUMO
The synthesis and optical characterisation of a series of green- and red-emitting cyanine and cyanine-styryl dyes is presented that were developed based on the cyanine-indole-quinolinium and based on the thiazole red type structure. For the green emitting fluorophores the quinolinium part was replaced by a pyridinium group. The bridge to the indole group was attached either to the 2-position or to the 4-position of the pyridinium moiety. For the red-emitting dyes the connection to the indole moiety is at the 4-position of the quinolinium part. In each set of dyes a methyl group at the indole-NH and/or a phenyl group at the 2-position of the indole part were introduced to tune the optical properties and photostability. Additionally, two dyes were modified with a cyano group to tune the photophysical properties and to enhance the photostabilities. The developed dyes show good photostabilities and bright green or red fluorescence intensities in the presence of DNA. Thus, these dyes represent important and promising candidates for fluorescent molecular imaging of nucleic acids inside living cells.
RESUMO
The photostability of cyanine-styryl dyes of the indole-quinolinium type can be significantly improved by structural variations while the excellent optical properties including the bright fluorescence in the presence of DNA can be maintained or even improved, too.