Electrocatalysis offers a means of electrochemical signal amplification, yet in DNA-based sensors, electrocatalysis has required highdensity DNA films and strict assembly and passivation conditions. Here, we describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low-density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low-density DNA films. Electrocatalysis with methylene blue that is covalently tethered to the DNA by a flexible alkyl chain linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of a single cytosine-adenine (CA) mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNAmediated charge transport. Electrocatalysis with hemoglobin is robust: It is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling.DNA charge transport | DNA sensors | mismatch detection
Metallic nanoparticles (MNPs) are prevalent in modern nanotechnologies due to their unique optical properties, chemical and photostability, and ease of manipulation. In particular, many recent advances have highlighted the importance of fundamentally understanding dynamic reconfiguration in MNP morphologies and compositions. Techniques to measure the shape of a single particle are lacking, however, often requiring immobilization, extensive numerical simulations, and irreversible alterations of the particle or its environment. In this work, we introduce “single-particle dynamic light scattering” (SP-DLS) as a far-field technique capable of analyzing the shape of individual, freely diffusing MNPs. Assuming symmetric-top rotors for MNPs and passively confining them to the focal volume of a dark-field microscope for long-term observation, we directly relate polarization dynamic fluctuations in the scattered light to the relative difference between the nondegenerate axes of individual particles. Our results show remarkable agreement with transmission electron microscopy analyses of the same population and allow for unprecedented measurements of the extent of prolate or oblate asphericity of nominally spherical MNPs in solution where the current implementation affords an asphericity detection limit of ∼2.5% assuming a 10% relative error. SP-DLS should serve as a powerful, nondestructive technique for characterizing the shapes of individual MNPs and other nanostructures.
Naturally split inteins drive the ligation of separately expressed polypeptides through a process called protein trans splicing (PTS). The ability to control PTS, so-called conditional protein splicing (CPS), has led to the development of tools to modulate protein structure and function at the post-translational level. CPS applications that utilize proximity as a trigger are especially intriguing as they afford the possibility to activate proteins in both a temporal and spatially targeted manner. In this study, we present the first proximity triggered CPS method that utilizes a naturally split fast splicing intein, Npu. We show that this method is amenable to diverse proximity triggers and capable of reconstituting and locally activating the acetyltransferase p300 in mammalian cells. This technology opens up a range of possibilities for the use of proximity triggered CPS.
Single-particle dynamic light scattering (SP-DLS) is a recently developed technique that uses dark-field illumination, active real-time three-dimensional single-particle tracking, and measurements of scattered photon polarizations to nonperturbatively evaluate the shapes of single, freely diffusing particles under the assumption of the particle having either prolate or oblate spheroid geometry. As originally developed, however, SP-DLS is incapable of unambiguously assigning either of these geometries to a single particle. In this contribution, we resolve this ambiguity by introducing a second experimental observablethe scattering spectrumso that both the scattering polarization and spectrum are simultaneously recorded and analyzed. We used numerical simulations of SP-DLS to characterize the performance of this new approach as well as the effects of key experimental parameters. We anticipate that the analyses presented here will not only form a straightforward guide for researchers seeking to optimize their own SP-DLS shape measurements but also serve as the basis for future studies of time-dependent reconfiguration in single nanostructures.
Recibido: 21/02/16 Evaluado: 22/03/16 * Parte de este escrito fue presentado como ponencia en la Primera Bienal de Infancias y Juventudes realizada en noviembre de 2013. El tema abordado corresponde a avances de la investigación "Escuela y conflicto armado en Colombia", financiada por la Universidad Santo Tomás.
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