Compositions – Inorganic luminescent compositions with organic...
Reexamination Certificate
2002-01-15
2004-08-03
Koslow, C. Melissa (Department: 1755)
Compositions
Inorganic luminescent compositions with organic...
C252S408100, C435S181000, C524S054000, C530S811000, C530S816000, C436S172000
Reexamination Certificate
active
06770220
ABSTRACT:
The invention relates to the composition, preparation and use of luminescent micro- and nanoparticles with long-lived luminescence. Said particles may be used either as internal standards for referencing fluorescence or phosphorescence signals (luminescence signals) or as markers for labeling and detecting biomolecules. Long-lived luminescent dyes are incorporated in an inert form into solid materials, i.e. shielded from the influence of chemical and biological substances in gaseous and aqueous samples. In this incorporated form, the photophysical properties of the dyes (spectral characteristics, luminescence decay time and luminescence anisotropy) remain unaffected by changing sample parameters.
The incorporating matrix selected is in particular compact inorganic materials or organic polymers which, due to their structure, exclude the uptake of biomolecules, small neutral molecules and also ionic substances. In particular, the interfering influence of molecular oxygen, an efficient fluorescence or phosphorescence quencher, on luminescence measurements is in this way eliminated or greatly reduced. The surface of said nano- and microparticles may be provided with reactive chemical groups, in order to make possible covalent coupling of biomolecules or/and luminescent indicator dyes. Furthermore, the surface may be provided with chemical groups in order to prevent the particles from aggregating.
Luminescence measurement is a very common method in biological and chemical analysis. Its attractiveness is due to its high sensitivity, versatility and also the elimination of radiation exposure by radioactive labeling reagents. In practice, luminescent markers distinguished by a high quantam yield are normally used. In most cases, the luminescence intensity of the luminescent marker is correlated with the sample parameter to be determined. Those determination methods are adversely affected by the fact that a multiplicity of factors interferes with the quantitative evaluation of luminescence intensity. Said factors may include firstly variations in the optical system (radiation intensity of the light source, detector sensitivity and transmission of the optical path), but also intrinsic optical properties of the sample (coloration or turbidity).
In order to eliminate or reduce said interfering influences, suitable methods for referencing the luminescence signals are required. WO 99/06821 (Klimant) describes a method for referencing luminescence signals, which is based on adding to the sample a luminescent reference dye which has similar (at best identical) spectral properties to the actual luminescent marker. In this way and in combination with frequence-modulated or time-resolved luminescence measurement, the intensity information is converted into a phase signal or a time-dependent parameter. In order to carry out correct referencing of the measurement signal in this way, inert luminescent reference standards are required, whose luminescence properties are not adversely affected by the sample parameters. Suitable for this purpose are, for example, phosphorescent inorganic solids such as, for example, Cr(III)-doped mixed oxides which can be admixed to the sample in powder form. On the other hand, it is also possible for this purpose to incorporate long-lived luminescent dyes into carriers made of organic or inorganic materials and admix the sample therewith.
Another type of interference of the quantitative evaluation of fluorescence intensity signals is the occurrence of intrinsic fluorescence in the sample. Natural samples such as blood or serum, in particular, can have a multiplicity of fluorescent substances. If the signal intensity of the fluorimetric assay is very low, intrinsic fluorescence may even render the measurement impossible. A widespread method for removing the actual luminescence signal from the unspecific background signal is to use luminescent dyes with long-lived emission as markers. It is possible, with the aid of time-resolved luminescence techniques, to separate by time the delayed measurement signal from the short-lived background fluorescence. This method uses mainly phosphorescent chelates of the rare earth metals (in particular those of europium or terbium). However, said dyes have the disadvantage that they can only be excited by UV light sources. Moreover, the chelates are often unstable when used in soluble form in aqueous systems, i.e. the ligands are lost. However, suitable long-lived markers are potentially also luminescent metal/ligand complexes, in particular those with ruthenium(II) as central atom. If these dyes are added in soluble form to aqueous systems, their luminescence is normally quenched by molecular oxygen, strong oxidants or reducers.
Furthermore, it is also possible, for example for determining the pH, the concentration or activity of ions or small molecules, to use luminescent indicators whose luminescence intensity depends on the concentration or activity of the parameter to be determined, for example an analyte or the pH, due to direct or indirect interaction with the parameter to be determined, for example due to reaction with an analyte or as transducer.
All methods mentioned absolutely require the photophysical properties of the luminescent dye to be unaffected by the sample parameters. These preconditions are not met if such dyes are added in dissolved form to the sample or contacted at least indirectly with the sample. Fluoresence or phosphoresence quenching by molecular oxygen and also oxidizing and reducing quenchers cause misinterpretations of the measurement signal.
In order to have available inert long-lived luminescent markers and luminescent dyes for referencing the luminescence intensity of luminescent indicators, the luminescent dyes have to be incorporated into solid materials so that they are incapable of interacting with the sample.
The present application describes both novel luminescent micro- and nanoparticles whose luminescence properties depend negligibly, if at all, on the sample composition, and methods for the preparation thereof. In addition, possible applications of the luminescent markers or luminescent dyes, present in the form of nano- and microparticles, for referencing the luminescence intensity of luminescent indicators are described.
The application therefore relates to luminescent, in particular phosphorescent, micro- and nanoparticles containing luminescent substances, for example metal/ligand complexes with long luminescence cay times, in a solid matrix so that they are shielded from ambient chemical parameters, for example a sample, and the luminescence properties of which, such as quantam yield, spectral characteristics, luminescence cay time or/and anisotropy, are essentially independent of the particular environment, for example the particular sample composition.
“Independents” in accordance with the subject application means that the dependence of the luminescence decay time and, where appropriate, further luminescence properties on the PO
2
and, where appropriate, other interfering substances in the environment of the luminescent dyes which are present in the particles of the invention and are at least in indirect contact with the sample is lower then the dependence of the luminescence decay time and, where appropriate, further luminescence properties of the corresponding dyes which are at least in indirect contact with the sample, without the inventive shielding.
Preferably, the luminescence lifetime of the luminescent dyes present in the particles of the invention is in an air-saturated environment at most 20%, particularly preferably at most 15% and most preferably at most 10% shorter than in an O
2
-free environment, in each case at room temperature. Without shielding, however, a reduction in the luminescence decay time by distinctly more than 80% is found in an air-saturated environment compared with an O
2
-free environment.
The luminescent metal/ligand complexes are preferably compounds of transition metals such as ruthenium(II), osmium(II), rhenium(I), iridium(III), platinum(II) and pa
Koslow C. Melissa
Presens Precision Sensing GmbH
Sutherland & Asbill & Brennan LLP
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