Molecular fluorophores, such as FITC and RPE, show a rapid decay understressing conditions. Aczon silica nanoparticles, thanks to the protective core / shell structure, protent the sensitive flourescent moleculesincreasing the fluorophores stability.
Considerable efforts have been made to improve immunofluorescence assays on both reagents and instruments. Often the time needed by the operator to focus the samples and take pictures results in the bleaching of the fluorescent molecules and the consequent signal intensity loss. In addition, the recent need of multiparametric analyses brought to the exploitation of multiple tandem dyes in flow cytometry. The main drawback of this kind of products, lays in the high instability of the acceptor molecules (usually cyanines).
AcZon nanoparticles are core/shell silica nanoparticles. The sensitive fluorescent molecules, covalently bounded inside the silica matrix, benefit of the silica shield effects. The external PEGylated shell allows the water solubility and the anchoring of targeting biomolecules such as antibodies or peptides. The increased stability of the nanoparticles-based fluorochromes has been shown under stressing conditions with respect of traditionally used molecular fluorochromes.
The nanoparticles used for the stability assessment experiment are NTB530 (max ex. 498; max em. 532nm) against the respective molecular fluorophore, fluorescein (max ex. 495nm; max em. 519nm), and NTB575 (max ex. 498; max em. 585nm) against the respective molecular fluorophore, phycoerythrin (max ex. 496nm; max em. 576nm). Molecular fluorophores and nanoparticles-based fluorophores have been embedded in a thin agarose gel matrix. The gel has been continuously irradiated by means of a mercury lamp under a microscope. Pictures have been taken at regular intervals (0, 20’, 40’, 60’) with a CCD camera. The fluorescence intensity has been analysed directly on the pictures with ImageJ software. The normalized fluorescence intensity data have been plotted on a Cartesian graph against exposure time (Fig.1). The results underline a dramatic decrease of the molecular fluorophores fluorescence intensity in the first 20 minutes of exposition which isn’t observed in the nanoparticles-based fluorophores.
The strength of the results is easily findable directly from the images in which, molecular fluorophores signal disappears in the early moments of the irradiation (Fig.2 and Fig.3).
Moreover, the higher number of fluorescent molecules included in each nanoparticle, increases the number of fluorophores for biomolecule unit, enhancing the signal with respect of the conventionally produced reagents (Fig.4). The advantages brought by this innovation are easy to be imagined. Thanks to the reactive moieties located on the external shell, AcZon silica nanoparticles may be conjugated with peptides and antibodies, increasing the reagents stability and, consequently, the sensitivity of immunofluorescence assays (Fig.5).
The increased stability and brightness brought by AcZon silica nanoparticles will improve the quality of immunofluorescence assays. These advantages are obtained thanks to the silica matrix in which molecular fluorophores are included. The employment of this new class of reagents allows the collection of higher quality images in fluorescence microscopy and better resolved populations analysis in flow cytometry.
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