The Experts below are selected from a list of 279 Experts worldwide ranked by ideXlab platform
Colette Mcdonagh - One of the best experts on this subject based on the ideXlab platform.
-
Fluorescent Cy5 silica nanoparticles for cancer cell imaging
Biosensing and Nanomedicine VIII, 2015Co-Authors: Claire O'connell, Robert Nooney, Macdara Glynn, Jens Ducrée, Colette McdonaghAbstract:Cancer is a leading cause of death worldwide, with metastasis responsible for the majority of cancer-related deaths. Circulating tumour cells (CTCs) play a central role in metastasis. Fluorescent silica particles (NPs), of diameter ~50 nm which contain a large concentration of Cy5 Dye molecules and are extremely bright, have been developed to detect these rare CTCs. Due to this brightness, the particles have superior performance compared to single Cy5 Dye molecule labels, for detecting cancer cells. Fluorescence measurements show that the NPs are almost 100 times brighter than the free Dye. They do not photo bleach as readily and, due to the biocompatible silica surface, they can be chemically modified, layer-by-layer, in order to bind to cells. The choice of these chemical layers, in particular the NP to antibody linker, along with the incubation period and type of media used in the incubation, has a strong influence on the specific binding abilities of the NPs. In this work, NPs have been shown to selectively bind to the MCF-7 cell line by targeting epithelial cellular adhesion molecule (EpCAM) present on the MCF-7 cell membrane by conjugating anti-EpCAM antibody to the NP surface. Results have shown a high signal to noise ratio for this cell line in comparison to a HeLa control line. NP attachment to cells was verified qualitatively with the use of fluorescence microscopy and quantitatively using image analysis methods. Once the system has been optimised, other Dyes will be doped into the silica NPs and their use in multiplexing will be investigated.
-
Enhancing the analytical performance of immunoassays that employ metal-enhanced fluorescence
Analytical and Bioanalytical Chemistry, 2010Co-Authors: Robert Nooney, Colette Mcdonagh, Ondrej Stranik, Andrew Clifford, Xavier Leguevel, Brian MaccraithAbstract:In this work, we used a model assay system (polyclonal human IgG-goat antihuman IgG) to elucidate some of the key factors that influence the analytical performance of bioassays that employ metal-enhanced fluorescence (MEF) using silver nanoparticles (NPs). Cy5 Dye was used as the fluorescent label, and results were compared with a standard assay performed in the absence of NPs. Two sizes of silver NPs were prepared with respective diameters of 60±10 and 149±16 nm. The absorption spectra of the NPs in solution were fitted accurately using Mie theory, and the dipole resonance of the 149-nm NPs in solution was found to match well with the absorption spectrum of Cy5. Such spectral matching is a key factor in optimizing MEF. NPs were deposited uniformly and reproducibly on polyelectrolyte-coated polystyrene substrates. Compared to the standard assay performed without the aid of NPs, significant improvements in sensitivity and in limit of detection (LOD) were obtained for the assay with the 149-nm NPs. An important observation was that the relative enhancement of fluorescence increased as the concentration of antigen increased. The metal-assisted assay data were analyzed using standard statistical methods and yielded a LOD of 0.086 ng/mL for the spectrally matched NPs compared to a value of 5.67 ng/mL obtained for the same assay in the absence of NPs. This improvement of ∼66× in LOD demonstrates the potential of metal-enhanced fluorescence for improving the analytical performance of bioassays when care is taken to optimize the key determining parameters.
-
Experimental and theoretical studies of the optimisation of fluorescence from near-infrared Dye-doped silica nanoparticles
Analytical and Bioanalytical Chemistry, 2009Co-Authors: Robert Nooney, Colette Mcdonagh, Ondrej Stranik, Ciara Mccahey, Xavier Le Guével, Brian MaccraithAbstract:There is substantial interest in the development of near-infrared Dye-doped nanoparticles (NPs) for a range of applications including immunocytochemistry, immuno-sorbent assays, flow cytometry, and DNA/protein micro-array analysis. The main motivation for this work is the significant increase in NP fluorescence that may be obtained compared with a single Dye label, for example Cy5. Dye-doped NPs were synthesised and a reduction in fluorescence as a function of Dye concentration was correlated with the occurrence of homo-Förster resonance energy transfer (HFRET) in the NP. Using standard analytical expressions describing HFRET, we modelled the fluorescence of NPs as a function of Dye loading. The results confirmed the occurrence of HFRET which arises from the small Stokes shift of near-infrared Dyes and provided a simple method for predicting the optimum Dye loading in NPs for maximum fluorescence. We used the inverse micelle method to prepare monodispersed silica NPs. The NPs were characterised using dynamic light scattering, UV spectroscopy, and transmission electron microscopy (TEM). The quantum efficiency of the Dye inside the NPs, as a function of Dye loading, was also determined. The fluorescent NPs were measured to be approximately 165 times brighter than the free Dye, at an optimal loading of 2% (w/w). These experimental results were in good agreement with model predictions.
-
Experimental and theoretical studies of the optimisation of fluorescence from near-infrared Dye-doped silica nanoparticles
Analytical and Bioanalytical Chemistry, 2009Co-Authors: Robert I. Nooney, Colette Mcdonagh, Ondrej Stranik, Ciara Mccahey, Xavier Le Guével, Brian D. MaccraithAbstract:There is substantial interest in the development of near-infrared Dye-doped nanoparticles (NPs) for a range of applications including immunocytochemistry, immunosorbent assays, flow cytometry, and DNA/protein microarray analysis. The main motivation for this work is the significant increase in NP fluorescence that may be obtained compared with a single Dye label, for example Cy5. Dye-doped NPs were synthesised and a reduction in fluorescence as a function of Dye concentration was correlated with the occurrence of homo-Förster resonance energy transfer (HFRET) in the NP. Using standard analytical expressions describing HFRET, we modelled the fluorescence of NPs as a function of Dye loading. The results confirmed the occurrence of HFRET which arises from the small Stokes shift of near-infrared Dyes and provided a simple method for predicting the optimum Dye loading in NPs for maximum fluorescence. We used the inverse micelle method to prepare monodispersed silica NPs. The NPs were characterised using dynamic light scattering, UV spectroscopy, and transmission electron microscopy (TEM). The quantum efficiency of the Dye inside the NPs, as a function of Dye loading, was also determined. The fluorescent NPs were measured to be approximately 165 times brighter than the free Dye, at an optimal loading of 2% ( w / w ). These experimental results were in good agreement with model predictions. Figure The change in nanoparticle fluorescence versus increased Dye loading modelled using homo-Förster resonance energy transfer.
-
Plasmonic enhancement of fluorescence for sensor applications
Photonic Crystal Materials and Nanostructures, 2004Co-Authors: Ondrej Stranik, Colette Mcdonagh, Brian D. MaccraithAbstract:It is well established that the presence of metallic surfaces or particles in the vicinity of a fluorophore can dramatically increase the radiative decay rate, and consequently the quantum efficiency, of the fluorophore. This effect, which depends on parameters such as metal particle size and fluorophore-particle separation, is manifest as a substantial enhancement in fluorescence emission intensity. This presentation will focus on optimisation strategies to maximise the enhancement for important applications such as fluorescence-based biochip platforms. Ordered arrays of metallic nano-islands were fabricated on a range of substrates by a process of natural lithography using monodisperse polystyrene nanospheres. The metal particle dimensions were tailored in order to match the plasmon resonance wavelength to the spectral absorption of the fluorophore. The fluorophore Cy5 Dye, which is widely used in optical immunoassays and has a medium quantum efficiency (~0.3), was used in this study of the plasmonic enhancement effect. The morphology of the metallic arrays was investigated using scanning electron microscope (SEM) and atomic force microscope (AFM). Absorption and emission spectroscopies were used to elucidate the enhancement effect and its dependence on metal island morphology. Results were correlated with existing theoretical models. The applicability of this important technique to sensor platforms, such as fluorescence-based biochips, will be discussed.
Andrea Rentmeister - One of the best experts on this subject based on the ideXlab platform.
-
Dual 5' Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions
Chemistry: A European Journal, 2017Co-Authors: Josephin Marie Holstein, Fabian Muttach, Stephan H. H. Schiefelbein, Andrea RentmeisterAbstract:The ability to detect and localize defined RNA strands inside living cells requires probes with high specificity, sensitivity, and signal-to-background ratio. To track low-abundant biomolecules, such as strands of regular mRNA, and distinguish fluorescence signal from the background after bioorthogonal reactions in cells, it is imperative to employ turn-on concepts. Here, we have presented a straightforward enzymatic approach to allow site-specific modification of two different positions on the 5′ cap of eukaryotic mRNA with either identical or different small functional groups. The approach relies on two methyltransferases and analogues of their natural co-substrate, and it can be extended to a three-enzyme cascade reaction for their in situ production. Subsequent labeling by using bioorthogonal click reactions provided access to double labeling with identical fluorophores or dual labeling with two different reporter groups, as exemplified by a Cy5 Dye, a FRET pair, and a fluorophore/biotin combination. Our dual-labeling strategy addresses the need for increased sensitivity and should improve the signal-to-background ratio after bioorthogonal reactions in cells.
-
Dual 5′ Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions
Chemistry (Weinheim an der Bergstrasse Germany), 2017Co-Authors: Josephin Marie Holstein, Fabian Muttach, Stephan H. H. Schiefelbein, Andrea RentmeisterAbstract:The ability to detect and localize defined RNA strands inside living cells requires probes with high specificity, sensitivity, and signal-to-background ratio. To track low-abundant biomolecules, such as strands of regular mRNA, and distinguish fluorescence signal from the background after bioorthogonal reactions in cells, it is imperative to employ turn-on concepts. Here, we have presented a straightforward enzymatic approach to allow site-specific modification of two different positions on the 5' cap of eukaryotic mRNA with either identical or different small functional groups. The approach relies on two methyltransferases and analogues of their natural co-substrate, and it can be extended to a three-enzyme cascade reaction for their in situ production. Subsequent labeling by using bioorthogonal click reactions provided access to double labeling with identical fluorophores or dual labeling with two different reporter groups, as exemplified by a Cy5 Dye, a FRET pair, and a fluorophore/biotin combination. Our dual-labeling strategy addresses the need for increased sensitivity and should improve the signal-to-background ratio after bioorthogonal reactions in cells.
Christoph Böttcher - One of the best experts on this subject based on the ideXlab platform.
-
Tubular J-aggregates of a new thiacarbocyanine Cy5 Dye for the far-red spectral region - a spectroscopic and cryo-transmission electron microscopy study.
Physical chemistry chemical physics : PCCP, 2018Co-Authors: Hans Von Berlepsch, Christoph BöttcherAbstract:The aggregation behavior of a phenol-substituted thiacarbocyanine Cy5 Dye (5-chloro-2-[5-[5-chloro-3-(4-sulfobutyl)-3H-benzothiazol-2-ylidene]-3-phenyl-penta-1,3-dienyl]-3-(4-sulfobutyl)-benzothiazol-3-ium hydroxide, inner salt, triethylammonium salt) in aqueous solution is investigated using steady-state absorption, linear dichroism, and fluorescence spectroscopies, as well as cryogenic transmission electron microscopy (cryo-TEM). By increasing the concentration, the Dye self-assembles in pure water into dimers and H-aggregates, the latter being uniform particles of ∼2.6 nm size. In the presence of NaCl, two different types of J-aggregates are observed depending on salt concentration (varied from 10 to 100 mM). At low salt concentration (10 mM) a J-aggregate of extended mono-layered sheets prevails, which disappears after a few days, whereas a second type of J-aggregate emerges. Generally, the latter dominates in matured solutions in particular at high salt concentration and seems to be the thermodynamically stable species. This J-aggregate shows three perpendicularly polarized absorption bands and fluoresces in the far-red at around 800 nm. The most intensive and very narrow (fwhm of 238 cm−1) absorption band is centered at 796 nm. Cryo-TEM reveals uniform nanotubes of ∼7 nm diameter and micrometer length. They represent the first tubular cyanine Dye J-aggregates that are active in the far-red. Moreover, the studied Dye is a prime example of cyanine Dyes showing two self-assembly pathways that lead to different species of J-aggregates with distinct optical and morphological properties.
-
H-Aggregates of an Indocyanine Cy5 Dye: Transition from Strong to Weak Molecular Coupling.
The journal of physical chemistry. B, 2015Co-Authors: Hans Von Berlepsch, Christoph BöttcherAbstract:The aggregation behavior of an Indocyanine Cy5 Dye (2-[5-[1,1-dimethyl-3-(4-sulfobutyl)-1,3-dihydro-benzo[e]indol-2-ylidene]-penta-1,3-dienyl]-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium hydroxide, inner salt, sodium salt) in aqueous solution is investigated using absorption and fluorescence spectroscopies, as well as cryogenic transmission electron microscopy (cryo-TEM). The Dye concentration is varied within a broad range from ∼1 μM to ∼10 mM. At moderate concentrations, typical H-aggregates are formed. After longer storage time, the absorption spectra of these solutions change dramatically. The characteristic blue-shifted absorption band at around 600 nm becomes replaced by a three-banded absorption spectrum, which spreads over a wide wavelength range of ∼600 up to 800 nm. However, at the highest Dye concentration and in the presence of ∼(10 to 30) mM NaCl, the three-banded spectrum is observed directly after preparation. The spectroscopic features can be ascribed to a structural transformation o...
Ondrej Stranik - One of the best experts on this subject based on the ideXlab platform.
-
Enhancing the analytical performance of immunoassays that employ metal-enhanced fluorescence
Analytical and Bioanalytical Chemistry, 2010Co-Authors: Robert Nooney, Colette Mcdonagh, Ondrej Stranik, Andrew Clifford, Xavier Leguevel, Brian MaccraithAbstract:In this work, we used a model assay system (polyclonal human IgG-goat antihuman IgG) to elucidate some of the key factors that influence the analytical performance of bioassays that employ metal-enhanced fluorescence (MEF) using silver nanoparticles (NPs). Cy5 Dye was used as the fluorescent label, and results were compared with a standard assay performed in the absence of NPs. Two sizes of silver NPs were prepared with respective diameters of 60±10 and 149±16 nm. The absorption spectra of the NPs in solution were fitted accurately using Mie theory, and the dipole resonance of the 149-nm NPs in solution was found to match well with the absorption spectrum of Cy5. Such spectral matching is a key factor in optimizing MEF. NPs were deposited uniformly and reproducibly on polyelectrolyte-coated polystyrene substrates. Compared to the standard assay performed without the aid of NPs, significant improvements in sensitivity and in limit of detection (LOD) were obtained for the assay with the 149-nm NPs. An important observation was that the relative enhancement of fluorescence increased as the concentration of antigen increased. The metal-assisted assay data were analyzed using standard statistical methods and yielded a LOD of 0.086 ng/mL for the spectrally matched NPs compared to a value of 5.67 ng/mL obtained for the same assay in the absence of NPs. This improvement of ∼66× in LOD demonstrates the potential of metal-enhanced fluorescence for improving the analytical performance of bioassays when care is taken to optimize the key determining parameters.
-
Experimental and theoretical studies of the optimisation of fluorescence from near-infrared Dye-doped silica nanoparticles
Analytical and Bioanalytical Chemistry, 2009Co-Authors: Robert I. Nooney, Colette Mcdonagh, Ondrej Stranik, Ciara Mccahey, Xavier Le Guével, Brian D. MaccraithAbstract:There is substantial interest in the development of near-infrared Dye-doped nanoparticles (NPs) for a range of applications including immunocytochemistry, immunosorbent assays, flow cytometry, and DNA/protein microarray analysis. The main motivation for this work is the significant increase in NP fluorescence that may be obtained compared with a single Dye label, for example Cy5. Dye-doped NPs were synthesised and a reduction in fluorescence as a function of Dye concentration was correlated with the occurrence of homo-Förster resonance energy transfer (HFRET) in the NP. Using standard analytical expressions describing HFRET, we modelled the fluorescence of NPs as a function of Dye loading. The results confirmed the occurrence of HFRET which arises from the small Stokes shift of near-infrared Dyes and provided a simple method for predicting the optimum Dye loading in NPs for maximum fluorescence. We used the inverse micelle method to prepare monodispersed silica NPs. The NPs were characterised using dynamic light scattering, UV spectroscopy, and transmission electron microscopy (TEM). The quantum efficiency of the Dye inside the NPs, as a function of Dye loading, was also determined. The fluorescent NPs were measured to be approximately 165 times brighter than the free Dye, at an optimal loading of 2% ( w / w ). These experimental results were in good agreement with model predictions. Figure The change in nanoparticle fluorescence versus increased Dye loading modelled using homo-Förster resonance energy transfer.
-
Experimental and theoretical studies of the optimisation of fluorescence from near-infrared Dye-doped silica nanoparticles
Analytical and Bioanalytical Chemistry, 2009Co-Authors: Robert Nooney, Colette Mcdonagh, Ondrej Stranik, Ciara Mccahey, Xavier Le Guével, Brian MaccraithAbstract:There is substantial interest in the development of near-infrared Dye-doped nanoparticles (NPs) for a range of applications including immunocytochemistry, immuno-sorbent assays, flow cytometry, and DNA/protein micro-array analysis. The main motivation for this work is the significant increase in NP fluorescence that may be obtained compared with a single Dye label, for example Cy5. Dye-doped NPs were synthesised and a reduction in fluorescence as a function of Dye concentration was correlated with the occurrence of homo-Förster resonance energy transfer (HFRET) in the NP. Using standard analytical expressions describing HFRET, we modelled the fluorescence of NPs as a function of Dye loading. The results confirmed the occurrence of HFRET which arises from the small Stokes shift of near-infrared Dyes and provided a simple method for predicting the optimum Dye loading in NPs for maximum fluorescence. We used the inverse micelle method to prepare monodispersed silica NPs. The NPs were characterised using dynamic light scattering, UV spectroscopy, and transmission electron microscopy (TEM). The quantum efficiency of the Dye inside the NPs, as a function of Dye loading, was also determined. The fluorescent NPs were measured to be approximately 165 times brighter than the free Dye, at an optimal loading of 2% (w/w). These experimental results were in good agreement with model predictions.
-
Plasmonic enhancement of fluorescence for sensor applications
Photonic Crystal Materials and Nanostructures, 2004Co-Authors: Ondrej Stranik, Colette Mcdonagh, Brian D. MaccraithAbstract:It is well established that the presence of metallic surfaces or particles in the vicinity of a fluorophore can dramatically increase the radiative decay rate, and consequently the quantum efficiency, of the fluorophore. This effect, which depends on parameters such as metal particle size and fluorophore-particle separation, is manifest as a substantial enhancement in fluorescence emission intensity. This presentation will focus on optimisation strategies to maximise the enhancement for important applications such as fluorescence-based biochip platforms. Ordered arrays of metallic nano-islands were fabricated on a range of substrates by a process of natural lithography using monodisperse polystyrene nanospheres. The metal particle dimensions were tailored in order to match the plasmon resonance wavelength to the spectral absorption of the fluorophore. The fluorophore Cy5 Dye, which is widely used in optical immunoassays and has a medium quantum efficiency (~0.3), was used in this study of the plasmonic enhancement effect. The morphology of the metallic arrays was investigated using scanning electron microscope (SEM) and atomic force microscope (AFM). Absorption and emission spectroscopies were used to elucidate the enhancement effect and its dependence on metal island morphology. Results were correlated with existing theoretical models. The applicability of this important technique to sensor platforms, such as fluorescence-based biochips, will be discussed.
Josephin Marie Holstein - One of the best experts on this subject based on the ideXlab platform.
-
Dual 5' Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions
Chemistry: A European Journal, 2017Co-Authors: Josephin Marie Holstein, Fabian Muttach, Stephan H. H. Schiefelbein, Andrea RentmeisterAbstract:The ability to detect and localize defined RNA strands inside living cells requires probes with high specificity, sensitivity, and signal-to-background ratio. To track low-abundant biomolecules, such as strands of regular mRNA, and distinguish fluorescence signal from the background after bioorthogonal reactions in cells, it is imperative to employ turn-on concepts. Here, we have presented a straightforward enzymatic approach to allow site-specific modification of two different positions on the 5′ cap of eukaryotic mRNA with either identical or different small functional groups. The approach relies on two methyltransferases and analogues of their natural co-substrate, and it can be extended to a three-enzyme cascade reaction for their in situ production. Subsequent labeling by using bioorthogonal click reactions provided access to double labeling with identical fluorophores or dual labeling with two different reporter groups, as exemplified by a Cy5 Dye, a FRET pair, and a fluorophore/biotin combination. Our dual-labeling strategy addresses the need for increased sensitivity and should improve the signal-to-background ratio after bioorthogonal reactions in cells.
-
Dual 5′ Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions
Chemistry (Weinheim an der Bergstrasse Germany), 2017Co-Authors: Josephin Marie Holstein, Fabian Muttach, Stephan H. H. Schiefelbein, Andrea RentmeisterAbstract:The ability to detect and localize defined RNA strands inside living cells requires probes with high specificity, sensitivity, and signal-to-background ratio. To track low-abundant biomolecules, such as strands of regular mRNA, and distinguish fluorescence signal from the background after bioorthogonal reactions in cells, it is imperative to employ turn-on concepts. Here, we have presented a straightforward enzymatic approach to allow site-specific modification of two different positions on the 5' cap of eukaryotic mRNA with either identical or different small functional groups. The approach relies on two methyltransferases and analogues of their natural co-substrate, and it can be extended to a three-enzyme cascade reaction for their in situ production. Subsequent labeling by using bioorthogonal click reactions provided access to double labeling with identical fluorophores or dual labeling with two different reporter groups, as exemplified by a Cy5 Dye, a FRET pair, and a fluorophore/biotin combination. Our dual-labeling strategy addresses the need for increased sensitivity and should improve the signal-to-background ratio after bioorthogonal reactions in cells.
