Anatoliy I. Dragan

Characterization of PicoGreen Interaction with dsDNA and the Origin of Its Fluorescence Enhancement upon Binding

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed.

Distance Dependence of Metal-Enhanced Fluorescence

In recent years both the mechanism and applications of metal-enhanced fluorescence (MEF) have attracted significant attention, yet many fundamental aspects of MEF remain unanswered or addressed. In this study, we address a fundamental aspect of MEF. Using fluorescein-labeled different length DNA scaffolds, covalently bound to silver nanodeposits, we have experimentally measured the distance dependence of the MEF effect. The enhanced fluorescence signatures, i.e., MEF, follow quite closely the theoretical decay of the near-field of the nanoparticles, calculated using finite difference time domain approaches. This implies that the mechanisms of MEF are partially underpinned by the magnitude and distribution of the electric field around near-field nanoparticles.

Metal-enhanced fluorescence: The role of quantum yield, Q0, in enhanced fluorescence

Metal-enhanced fluorescence has attracted enormous research and commercial interest in recent years, due to the ability to significantly enhance fluorescence signatures in the near-field as well as protect fluorophores against photobleaching. In this article, we address one of the major unresolved questions, whether far-field fluorophore quantum yield, Q0, has a direct relationship to fluorescence enhancement factors in metal-enhanced fluorescence.

Sialyl residues modulate LPS-mediated signaling through the Toll-like receptor 4 complex.

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation.

Metal-enhanced fluorescence from silver-SiO2-silver nanoburger structures.

Multilayers of nanoburger structures of silver island films-SiO(2)-silver island films (SIFs-SiO(2)-SIFs) were used as substrates to study the fluorescence of close-proximity fluorophores. Compared to single-layered SIFs, multilayer nanoburgers exhibit several distinctive properties including a significantly enhanced fluorescence intensity, decreased lifetimes, and increased fluorphore photostability by simply varying the dielectric layer thickness while the SIF layer is kept constant. Finite-difference time-domain (FDTD) calculations show that the maximum electric field intensity can be tuned by varying the distance between the silver particles. Enhanced fluorescence emission coupled with a reduced fluorophore lifetime suggests that both an electric field and plasmon-coupling component are the underlying mechanisms for nanoburger-based metal-enhanced fluorescence (MEF). This tunable multilayer nanoburger structure holds great potential for applications in biology, microscopy, imaging, and biomedical research, given the current uses of MEF.

Fluorescence-based Broad Dynamic Range Viscosity Probes

We introduce two new fluorescent viscosity probes, SYBR Green (SG) and PicoGreen (PG), that we have studied over a broad range of viscosity and in collagen solutions. In water, both dyes have low quantum yields and excited state lifetimes, while in viscous solvents or in complex with DNA both parameters dramatically (300–1000-fold) increase. We show that in log-log scale the dependence of the dyes’ quantum yield vs. viscosity is linear, the slope of which is sensitive to temperature. Application of SG and PG, as a fluorescence-based broad dynamic range viscosity probes, to the life sciences is discussed.

Mixed-metal substrates for applications in metal-enhanced fluorescence

Over the last decade Metal-Enhanced Fluorescence (MEF) has emerged as the next generation of fluorescence spectroscopy, i.e. near-field fluorescence. However, in contrast to our collective knowledge and understanding of classical far-field fluorescence, we know relatively little. MEF is a consequence of the near-field interactions of fluorophores (dipoles) with the surface plasmons generated in plasmon supporting materials, where the optical properties of the metal afford for a wavelength dependence of MEF. In this paper we show that we are not limited to the properties of the individual metals for MEF, but in fact, surface deposits of mixed metals can create new dephased plasmon resonance bands, not present in the individual metals themselves. Subsequently, mixed metal substrates (MMS) offer significant opportunities for the multifarious and forever growing applications of MEF.

5-Color Multiplexed Microwave-Accelerated Metal-Enhanced Fluorescence: Detection and Analysis of Multiple DNA Sequences from within one Sample Well within a Few Seconds

We present a potentially highly sensitive and selective bio-assay for the potential detection of any five different DNA sequences from one sample in one well. The assay is based on a DNA “rapid catch and signal” (DNA-RCS) technology developed for the detection of different DNA sequences from a sample well area. Our signal amplification utilizes the metal-enhanced fluorescence (MEF) of dyes attached to the probe-DNAs, which hybridizes with the pre-formed mixture of anchor-DNA scaffolds on silver island films (SiFs). Low-power microwave irradiation accelerates both the formation of the anchor-DNA scaffold on the SiF-surface and anchor/probe DNA hybridization, i.e. “rapid catch” of target DNAs from a bulk solution, decreasing the assay run time from hours to only a few seconds. Localization of signaling dye-labels close to the SiFs make them extremely photostable, which allows for collecting/integrating the signal over a long time period. To demonstrate a 5 color DNA assay (5-plex) we have used a range of readily available Alexa™ dyes. Advantages and perspectives of the RCS-technologies ability to detect 5 different DNA sequences from within one plate-well are discussed.