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
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.
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.
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.
It is an established understanding that the electromagnetic contribution (plasmon-mediated enhancement of a laser and scattered local electromagnetic fields) is the main actor in surface enhanced Raman scattering (SERS), with the so-called chemical (molecule-related) contribution assuming only, if any, a supporting role. The conclusion of our comprehensive experimental resonant study of a broad range of nanosphere lithography based metallic substrates, with covalently attached 4-mercaptobenzoic acid monolayers used as a probe (standard molecules that are non-resonant in solution), is that this accepted understanding needs to be revised. We present a detailed resonant SERS study of metal-film-over-nanosphere (MFON) substrates that is done by both scanning the laser wavelength and tuning the plasmon response through the nanosphere diameter, which is varied from 500 to 900 nm. Far and local field properties are characterized through measures of optical reflectivity and SERS efficiency, respectively, and are supported by numerical simulations. We demonstrate that SERS intensity depends indeed on the electromagnetic mechanism, determined by the plasmonic response of the system, but we observe that it is also strongly defined by a chemical resonant contribution related to a metal-to-ligand electronic transition of the covalently bound probe molecule. Optimum amplification occurs when the plasmon modes intersect with the ligand-to-metal chemical resonance, contributing synergically both mechanisms together. Quite notably, however, the largest SERS signal is observed when the laser is tuned with the metal-to-ligand transition, and typically does not follow the wavelength dependence of the plasmon modes when varying the nanosphere size. The same general trend is observed for other nanosphere lithography based substrates, including sphere segment void cavities and hexagonally ordered triangular nanoparticles, using either Ag or Au as the plasmonic metal, and also with a commercial substrate (Klarite). Interestingly, this extensive comparative investigation shows in addition that MFON substrates are significantly better than these other studied plasmonic substrates in terms of Raman intensity and homogeneity. We conclude that a deep understanding of both electromagnetic and chemical mechanisms is necessary to fully exploit these substrates for analytical applications.
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