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Alexa Fluor 488

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Juan G. Santiago – One of the best experts on this subject based on the ideXlab platform.

  • Detection of 100 aM Fluorophores using a high-sensitivity on-chip CE system and transient isotachophoresis.
    Analytical chemistry, 2007
    Co-Authors: Byoungsok Jung, Yonggang Zhu, Juan G. Santiago
    Abstract:

    We present a highly sensitive capillary electrophoresis (CE) assay that combines transient, single-interface on-chip isotachophoresis (ITP) and a laser-induced confocal Fluorescence detection setup. We performed experimental parametric studies to show the effects of microscope objective specifications and intensity of excitation laser on optimization of a high-sensitivity on-chip CE detection system. Using the optimized detection system, single-molecule detection of Alexa Fluor 488 was demonstrated, and signal data were validated with autocorrelation analysis. We also demonstrated a separation and detection of 100 aM Fluorophores (Alexa Fluor 488 and bodipy) in a fast assay using a high-sensitivity on-chip CE detection system and an ITP/CE protocol with no manual buffer exchange steps. This is, to the knowledge of the authors, the highest electrophoretic separation sensitivity ever reported.

  • On-chip millionfold sample stacking using transient isotachophoresis.
    Analytical chemistry, 2006
    Co-Authors: Byoungsok Jung, Rajiv Bharadwaj, Juan G. Santiago
    Abstract:

    We present a simple and robust isotachophoresis (ITP) method that can be integrated with microchip-based capillary electrophoresis (CE) devices to achieve millionfold sample stacking. We performed an experimental parametric study to show the effects of initial sample ion concentration, leading ion concentration, and trailing ion concentration on ITP stacking. We also discuss the usefulness and limitations of a simple one-dimensional nondispersive model and a scaling analysis for dispersion rate. We found that a single-column ITP configuration together with electroosmotic flow suppression and high leading ion concentration provide high-performance ITP and can be integrated readily with CE separation. We demonstrated detection of trace of 100 fM Alexa Fluor 488 (signal-to-noise ratio of 11) with a concentration increase of a factor of 2 × 106. Application of our ITP/CE protocol to the stacking and separation of negatively charged Fluorescent tracers (Alexa Fluor 488 and bodipy) resulted in a concentration i…

Raoul Kopelman – One of the best experts on this subject based on the ideXlab platform.

Byoungsok Jung – One of the best experts on this subject based on the ideXlab platform.

  • Detection of 100 aM Fluorophores using a high-sensitivity on-chip CE system and transient isotachophoresis.
    Analytical chemistry, 2007
    Co-Authors: Byoungsok Jung, Yonggang Zhu, Juan G. Santiago
    Abstract:

    We present a highly sensitive capillary electrophoresis (CE) assay that combines transient, single-interface on-chip isotachophoresis (ITP) and a laser-induced confocal Fluorescence detection setup. We performed experimental parametric studies to show the effects of microscope objective specifications and intensity of excitation laser on optimization of a high-sensitivity on-chip CE detection system. Using the optimized detection system, single-molecule detection of Alexa Fluor 488 was demonstrated, and signal data were validated with autocorrelation analysis. We also demonstrated a separation and detection of 100 aM Fluorophores (Alexa Fluor 488 and bodipy) in a fast assay using a high-sensitivity on-chip CE detection system and an ITP/CE protocol with no manual buffer exchange steps. This is, to the knowledge of the authors, the highest electrophoretic separation sensitivity ever reported.

  • On-chip millionfold sample stacking using transient isotachophoresis.
    Analytical chemistry, 2006
    Co-Authors: Byoungsok Jung, Rajiv Bharadwaj, Juan G. Santiago
    Abstract:

    We present a simple and robust isotachophoresis (ITP) method that can be integrated with microchip-based capillary electrophoresis (CE) devices to achieve millionfold sample stacking. We performed an experimental parametric study to show the effects of initial sample ion concentration, leading ion concentration, and trailing ion concentration on ITP stacking. We also discuss the usefulness and limitations of a simple one-dimensional nondispersive model and a scaling analysis for dispersion rate. We found that a single-column ITP configuration together with electroosmotic flow suppression and high leading ion concentration provide high-performance ITP and can be integrated readily with CE separation. We demonstrated detection of trace of 100 fM Alexa Fluor 488 (signal-to-noise ratio of 11) with a concentration increase of a factor of 2 × 106. Application of our ITP/CE protocol to the stacking and separation of negatively charged Fluorescent tracers (Alexa Fluor 488 and bodipy) resulted in a concentration i…

Chiwan Koo – One of the best experts on this subject based on the ideXlab platform.

  • A portable and high-sensitivity optical sensing system for detecting Fluorescently labeled enterohaemorrhagic Escherichia coli Shiga toxin 2B-subunit.
    PloS one, 2020
    Co-Authors: Jeongtae Kim, Jun-young Park, Young-jun Park, Seo-young Park, Moo-seung Lee, Chiwan Koo
    Abstract:

    We developed a stand-alone, real-time optical detection device capable of reading Fluorescence intensities from cell samples with high sensitivity and precision, for use as a portable Fluorescent sensor for sensing Fluorescently labeled enterohemorrhagic Escherichia coli (EHEC) Shiga toxins (Stxs). In general, the signal intensity from the Fluorescently labeled Stxs was weak due to the small number of molecules bound to each cell. To address this technical challenge, we used a highly sensitive light detector (photomultiplier tube: PMT) to measure Fluorescence, and designed a portable optical housing to align optical parts precisely; the housing itself was fabricated on a 3D printer. In addition, an electric circuit that amplified PMT output was designed and integrated into the system. The system shows the toxin concentration in the sample on a liquid crystal display (LCD), and a microcontroller circuit is used to read PMT output, process data, and display results. In contrast to other portable Fluorescent detectors, the system works alone, without any peripheral computer or additional apparatus; its total size is about 17 × 13 × 9 cm3, and it weighs about 770 g. The detection limit was 0.01 ppm of Alexa Fluor 488 in PBS, which is ten thousand times lower than those of other smartphone-based systems and sufficiently sensitive for use with a portable optical detector. We used the portable real-time optical sensing system to detect Alexa Fluor 488-tagged Stx2B-subunits bound to monocytic THP-1 cells expressing the toxin receptor globotriaosylceramide (Gb3). The device did not detect a signal from Gb3-negative PD36 cells, indicating that it was capable of specifically detecting Stxs bound to cells expressing the toxin receptor. Following the development of a rapid and autonomous method for Fluorescently tagging cells in food samples, the optical detection system described here could be used for direct detection of Shiga toxins in food in the field.

James P. Sumner – One of the best experts on this subject based on the ideXlab platform.

  • Cu+- and Cu2+-sensitive PEBBLE Fluorescent nanosensors using DsRed as the recognition element
    Sensors and Actuators B: Chemical, 2006
    Co-Authors: James P. Sumner, Nissa Westerberg, Andrea K. Stoddard, Carol A. Fierke, Raoul Kopelman
    Abstract:

    Abstract The red Fluorescent protein (DsRed) has typically been used as either a Fluorescent tag or FRET acceptor, but here, we detail its use as a recognition element for Cu2+ and Cu+ in a Fluorescent photonic explorer for bioanalysis with biologically localized embedding (PEBBLE) nanosensor. DsRed and the reference dye Alexa Fluor 488 have been encapsulated within a polyacrylamide matrix by a microemulsion polymerization process, to produce spherical, ratiometric, 80 nm-sized sensors, which allow for spatially resolved real-time measurements. These nanobiosensors have excellent selectivity and sensitivity towards copper ions, with a detectable range in the nanomolar (ppb) regime, even in the presence of other divalent and heavy metal ions. The nanosensors are both photostable and reversible, which allows for continuous monitoring.

  • Alexa Fluor 488 as an iron sensing molecule and its application in pebble nanosensors
    Analyst, 2005
    Co-Authors: James P. Sumner, Raoul Kopelman
    Abstract:

    Molecular Probes’ Alexa Fluor dyes are generally used for biological labeling because of their ideal Fluorescent properties, but here we detail Alexa Fluor 488‘s nanomolar sensitivity to free iron. Furthermore, the dye has been encapsulated into a polymer nanosphere by a microemulsion method, producing <100 nm particles. These nanosensors, PEBBLEs (Probe Encapsulated By Biologically Localized Embedding) have micromolar sensitivity and are non-responsive to other metal ions of biological interest.

  • Alexa Fluor 488 as an iron sensing molecule and its application in PEBBLE nanosensors
    The Analyst, 2005
    Co-Authors: James P. Sumner, Raoul Kopelman
    Abstract:

    Molecular Probes’ Alexa Fluor dyes are generally used for biological labeling because of their ideal Fluorescent properties, but here we detail Alexa Fluor 488‘s nanomolar sensitivity to free iron. Furthermore, the dye has been encapsulated into a polymer nanosphere by a microemulsion method, producing