Chemistry: analytical and immunological testing – Involving producing or treating antigen or hapten – Producing labeled antigens
Patent
1998-06-18
2000-10-24
Ceperley, Mary E.
Chemistry: analytical and immunological testing
Involving producing or treating antigen or hapten
Producing labeled antigens
435 6, 436531, 436800, 436817, 5303913, 530402, 530408, 548427, G01N 33533, C07D20956
Patent
active
061366129
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a new class of fluorescent dyes and their valence tautomers belonging to the sulfo benz[e]indocyanine family. The instant invention also relates to the synthesis of the new class of fluorescent dyes belonging to the sulfo benz[e]indocyanine family. The new fluorescent dyes can be excited using powerful yet inexpensive light emitting diodes and diode lasers, they exhibit good water solubility, and can be attached or conjugated to a wide variety of molecules or surfaces for labeling purposes. The new fluorescent dyes are particularly useful in techniques such as immunoassays, DNA probes, high pressure liquid chromatography (HPLC), capillary electrophoresis, fluorescence polarization, total internal reflection fluorescence (T.I.R.F.), flow cytometry, DNA sequencing, and optical sensors.
BACKGROUND OF THE INVENTION
The use of fluorescence technology has become widespread in the areas of clinical chemistry, i.e., laboratory testing and the medical diagnostic areas. The technology is particularly effective for making very sensitive and specific test determinations, competing effectively in many areas with radioimmunoassays and enzymatic immunoassays.
The phenomenon of fluorescence occurs when a molecule or atom is bombarded with light of given wavelengths; namely, the conversion of that light to an emission of light of a different wavelength. In macroscopic terms, the conversion is instantaneous, but in real terms the finite time differences between the absorption of the light by the molecule and the time interval during which the emitted light is given off is a measure of the characteristics of the bodies being measured.
The process of fluorescence starts with the absorption of light photons by atoms or molecules. The frequency of light absorption varies with the atom or molecule involved.
Fluorescent molecules in any specific environment have two characteristic spectra. The first, the so-called excitation spectrum, is represented by a series of wavelengths of light which are absorbed by the molecule with differing efficiencies. That is, out of a possible number of existing wavelengths which may be absorbed by the molecule to cause fluorescence, usually one of these will be absorbed at a greater level. Most atoms or molecules that absorb light convert this light energy into heat, but a few emit light or "fluoresce" at a lower light frequency. Photon absorption occurs rapidly in about 10.sup.-15 seconds. If the light excitation is abruptly interrupted, as with a very short pulse of light, photon light emission in the second spectrum will decay rapidly with a time constant that depends on the atom or molecule involved. The range of decay times is usually between 10.sup.-10 to 10.sup.-6 seconds (0.1 to 1000 nanoseconds). The intensity of the emission spectrum is directly proportional to the intensity of the exciting light.
It happens also that the intensity of the emitted light is also directly proportional to the concentration of the fluorescent molecules in the sample. It thus can be seen that a very sensitive technique for measuring the concentration of a fluorescent body can be evolved by controlling the intensity of the exciting light and other physical constants of the measuring system.
The analytical value of fluorescence decay time measurement arises from the fact that each atom or molecule has its own distinctive rate of decay. Each atom or molecule is excited at a different frequency and emits light only at a particular emission wavelength.
Analytical probes having fluorescent labels are valuable reagents for the analysis and separation of molecules and cells. Specific applications include: (1) identification and separation of subpopulations of cells in a mixture of cells by the techniques of fluorescence flow cytometry, fluorescence-activated cell sorting, and fluorescence microscopy; (2) determination of the concentration of a substance that binds to a second species (e.g., antigen-antibody reactions) in the technique of fluorescence immunoass
REFERENCES:
patent: 5268486 (1993-12-01), Waggoner et al.
patent: 5453505 (1995-09-01), Lee et al.
Caputo Giuseppe
Cassullo Mariacristina
Ciana Leopoldo Della
Grignani Andrea
Ceperley Mary E.
Sorin Biomedica Cardio S.p.A.
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