NTB520 are core-shell dye doped silica nanoparticles synthesized through a one-pot, two-steps reaction, deriving from Stöber synthesis, known as micelle-assisted method. All the reagents are mixed together in a solution of water and n-butanol to allow the creation of micelles from the surfactant. All the hydrophobic reagents used in the reaction, spontaneously arrange inside the micelles.
Thanks to the addition of ammonia and silane precursor, the base-catalysed hydrolysis of all the trialkoxysilanes takes place. The variation of surfactant characteristics affects the nanoparticles size. The initial mixture contains 2 different hydrophobic fluorophores arranging inside the micelles and becoming part of the nanoparticle, thanks to the covalent modification with a trialkoxysilane group: as part of the structure, dyes are not released over time. Even without a covalent binding among the 2 fluorophores, they can generate an efficient fluorescence resonance energy transfer (FRET).
The number of fluorophores is modulated to obtain the maximum efficiency of the cascade transfer without inducing the self-quenching phenomenon, thanks to these devices NTB520 reach over 90% of FRET efficiency. NTB520 has the same excitation and emission wavelengths of the commercial dyes FITC and AlexaFluor®488 The analyses on peripheral whole blood with anti CD4 conjugates with 3 different typologies of fluorophores, highlighted a good discrimination among negative and positive populations in each of the three cases. The test performed comparing the 3 conjugates at the same concentration, shows differences in the fluorescence intensity (figure 1).
Figure 1: Comparison among the three different conjugates at the same concentration
The higher fluorescence intensity of the positive population, due to the use of NTB520 instead of traditional fluorophores, is related to a lower fluorescence level of the negative population, increasing once more the signal-to-noise ratio.
The conjugation of fluorescent silica nanoparticles (like NTB520) to antibodies, combines the properties of nanoparticles to the inherent properties of antibodies such as the ability to specifically recognize antigens. Thanks to the isolation of the sensitive fluorescent molecules from the external, interfering, environment, the signal provided by this new class of fluorophores is more stable over time. This isolation solves another issue due to the non-specific binding of fluorophores to specific cellular populations such as monocytes, resulting in a reduced background.
The concentration capability allows the inclusion of more fluorophores in each nanoparticle, permitting the binding of more fluorescent molecules for single signalling unit (e.g. antibody) giving an enhanced fluorescence intensity (a decade respect of FITC and half a decade respect of AlexaFluor®488). To confirm the superiority of nanoparticles-based reagents respect of those using conventional fluorophores, the stain index has been calculated for all the conjugates (data not shown).
The stain index is the difference between the mean fluorescence of the positive population minus the central tendency of the negative population. This is divided by twice the standard deviation. Thanks to this measure, the fluorescence intensity of a fluorophore is normalized against all possible factors affecting it and can be compared among different fluorophores.The stain index of NTB520 resulted higher than those of other fluorophores.
These results confirm fluorescent silica nanoparticles as a promising tool for biomedical applications thanks to their high sensitivity, enhanced stability and reduced background noise. The stain index in flow cytometry of NTB520 conjugates results higher than those of other fluorophores. These results confirm fluorescent silica nanoparticles as a promising tool for biomedical applications thanks to their high sensitivity, enhanced stability and reduced background noise.