Experimental and theoretical studies of the optimisation of fluorescence from near-infrared dye-doped silica nanoparticles.
ABSTRACT 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.
- SourceAvailable from: Hassan Badrane[show abstract] [hide abstract]
ABSTRACT: Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.Analytical Biochemistry 12/2004; 334(1):135-44. · 2.58 Impact Factor
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ABSTRACT: The use of diode laser-induced fluorescence (DIO-LIF) detection in the field of capillary electrophoresis (CE) is examined. A simple but sensitive detection system was constructed. The performance of the system was evaluated with respect to design factors and its sensitivity was compared with the theoretically achievable sensitivity. To enhance the applicability of direct DIO-LIF detection in CE, a derivatization method for amines was developed. A red-absorbing label, consisting of a dicarbocyanine fluorophore with a succinimidyl ester functionality, was synthesized for this purpose. After derivatization of 1 x 10(-6) M glycine, a detection limit of 0.1 amol was observed for the labeled glycine. Similar detection limits were observed for other amino acids. To show that derivatization preserves the separation efficiency of CE for the analytes examined, 18 amino acids and tyramine were separated with micellar electrokinetic chromatography after labeling. In addition, even labeled peptides, including structurally related enkephalin-type compounds, were separated from each other with zone electrophoresis. To test the applicability of the derivatization method to biological samples, tyramine was determined in urine before and after the consumption of cheese.Journal of Chromatography 09/1995; 708(2):309-21. · 4.61 Impact Factor
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ABSTRACT: A new molecular conjugation method has been developed to label biomolecules with optically stable metalorganic luminophores, such as tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy), which are otherwise not possible for direct linking with the biomolecules. Unique biochemical properties of the biomolecule can, thus, be associated with photostable luminophores. This opens a general way to conjugate desired biomolecules using a sensitive signal transduction method. It also promotes the application of excellent luminescent materials, especially those based on photostable metalorganic luminophores, in biochemical analysis and biomolecular interaction studies. The conjugation method is based on uniform luminophore-doped silica (LDS) nanoparticles (63 +/- 4 nm). These nanoparticles have been prepared using a water-in-oil (W/O) microemulsion method. The controlled hydrolysis of tetraethyl orthosilicate (TEOS) in W/O microemulsion leads to the formation of monodisperse LDS nanoparticles. The luminophores are doped inside the nanoparticles, and the particle's silica surfaces can be used to covalently bind with biomolecules. The luminophores are well-protected from the environmental oxygen when they are doped inside the silica network. As an example, we used an antibody for leukemia cell recognition. The antibody was first immobilized onto the luminophore-doped nanoparticle through silica chemistry and then was used for leukemia cell identification by an optical microscopy imaging technique. The leukemia cells were identified easily, clearly, and with high efficiency using these antibody-coated nanoparticles. The advantages of using small, uniform luminophore-doped nanoparticles are discussed.Analytical Chemistry 11/2001; 73(20):4988-93. · 5.70 Impact Factor