Fluorescence energy transfer in particles

Details Fluorescence energy transfer in particles Fluorescence energy transfer in particles

1994-07-12

2001-05-29

Ceperley, Mary E. (Department: 1641)

Chemistry: analytical and immunological testing

Involving producing or treating antigen or hapten

Producing labeled antigens

C435S006120, C436S525000, C436S527000, C436S528000, C436S531000, C436S534000, C530S389200, C530S391300, C530S811000, C530S812000, C530S815000, C530S810000, C530S402000

Reexamination Certificate

active

06238931

ABSTRACT:

FIELD OF THE INVENTION
This invention relates generally to labels and methods for the detection or visualization of analytes and more specifically to fluorescent latex particles which incorporate fluorescence energy transfer for the detection of analytes in immunoassays.
BACKGROUND
Various methodologies are available for the visualization of cells or molecules in cells and for the measurement of analyte concentrations in fluids. Fluorescence microscopy utilizes fluorescent dyes, generally connected to specific probes, such as antibodies, for the localization of proteins and complexes in cells. For the measurement of analyte concentrations, immunoassays have become popular over the last 40 years because of the specificity of antibodies toward the analyte or target ligand. Radioimmunoassays were developed because the high specific activity of the radionuclide allowed measurement of very low concentrations of analyte. However, because of the concerns for the environment and human health, the use of radionuclides in immunoassays is becoming less popular. The use of enzymes in immunoassays to amplify a signal has been a very important advance in the field of immunoassays because their use does not involve environmental or human health hazards or risks. Enzyme-linked immunoassays, however, can be problematic because the activity of the enzyme is temperature dependent and the instability of the enzyme or the substrates can result in inaccurate quantitation of the target ligand. Still other immunoassays monitor fluorescence as the signal, with or without enzymes, for the measurement of analyte concentrations.
The characteristics of the fluorescent dyes are very important when quantifying analyte concentrations in biological fluids. For example, when the biological fluid is blood, serum or plasma, the intrinsic fluorescence of the fluid precludes the use of many dyes. These biological fluids generally have fluorescence emissions up to 600 nm when exciting at various wavelengths above 200 nm. The fluorescent signal is measured by a fluorometer which is tuned to excite the fluorescent molecule at a specific wavelength and to measure the emission of fluorescence at another wavelength. The difference in the excitation and emission wavelengths is referred to as the Stokes shift. To achieve the most sensitive measurement, the emission wavelength of the sample should not interfere with the emission of the dye. Also, the Stokes shift should be as large as possible so that the excitation light is not seen by the detector as noise. Where the Stokes shift is not large, filters or monochromators can be utilized in the fluorometer to exclude light near the emission wavelength; however, the use of filters decreases the yield of light reaching the detector and generally one circumvents this problem of light loss by the use of high intensity lamps. Thus, to avoid problems associated with small Stokes shifts and dyes which emit near the intrinsic emission of the biological fluid, a sophisticated instrument is generally built. With the advent of near-patient diagnostics in hospitals, instruments which are used for the diagnostics will become more portable and simpler to use. Therefore, there is a need for portable, simple fluorometers which can assess fluorescence in an immunoassay for the detection of analytes in biological samples.
Another problem associated with the assay of analytes in fluids or the visualization of cellular components with an intrinsic fluorescence is that of selection of the dye which is utilized as the label. The dye is generally chosen for its brightness (the product of fluorescence quantum yield and extinction coefficient) since a certain sensitivity in the assay or the visualization technique is required. However, the selection of the dye used as the label is limited when the sample has an intrinsic fluorescence because the instrument may not be capable of distinguishing sample fluorescence from dye fluorescence.
The current invention provides a methodology for the development of amplified fluorescent label systems which can be tuned to specific excitation and emission wavelengths. In addition, the methodology teaches improved methods for incorporation of dyes into particles to minimize fluorescence quenching and to maximize fluorescence intensities of the dye molecules in the particles. The novel dye systems can be utilized for the quantitation of analytes in fluids, and in particular, in biological fluids. The novel dye systems can be tuned to specific exciting and emitting wavelengths so that low current sources, such as light emitting diodes and laser diodes, and detectors, such as photo diodes, and the like, can be used in the manufacture of fluorometers which can be battery powered and portable, for use, for example, in immunoassays dedicated to near-patient diagnostics.
SUMMARY OF THE INVENTION
This invention relates to novel particles which exhibit fluorescence energy transfer (singlet-singlet energy transfer). These novel particles can be tuned to specific excitation and emission wavelengths to accommodate a wide variety of assay or visualization systems. In yet another aspect of the invention, the methodology teaches improved methods for incorporation of dyes into particles to minimize fluorescence quenching and to maximize fluorescence intensities of the dye molecules in the particles through the use of different dye molecules which possess the same or very similar excitation and emission wavelengths.
In a first aspect, the invention concerns particles that comprise an energy donor as a first component and an energy acceptor as a second component positioned in a particle at an energy exchanging distance from one another, the two components having a Stokes shift of greater than or equal to 50 nm, and the particle having bound on its surface, a protein, polypeptide, nucleic acid, nucleotide or protein containing ligand analogue. In certain embodiments, the particles also comprise at least one additional fluorescent dye as a third component that exhibits in the particle approximately the same excitation and emission wavelengths as the second component. In preferred embodiments, the particles are latex particles.
In another aspect, the invention features particles comprising an energy donor as a first component and a fluorescent dye as a second component positioned in a particle at an energy exchanging distance from one another, the two components having a Stokes shift of greater than or equal to 50 nm, and either the first or second components being phthalocyanine or naphthalocyanine. In certain embodiments, the particles also comprise at least one additional fluorescent dye as a third component that exhibits in the particle approximately the same excitation and emission wavelengths as the second component. In preferred embodiments, the particles are latex particles.
In particularly preferred embodiments, the first component is phthalocyanine and the second component is naphthalocyanine; the first component is styryl and the second component is phthalocyanine; the first component is styryl and the second component is naphthalocyanine; the first component is phenylbutadienyl and the second component is phthalocyanine; the first component is phenylbutadienyl and the second component is naphthalocyanine; the first component is phenylhexatrienyl and the second component is phthalocyanine; the first component is phenylhexatrienyl and the second component is naphthalocyanine; the first component is porphine and the second component is phthalocyanine; the first component is porphine and the second component is naphthalocyanine; the first component is a carbocyanine dye and the second component is phthalocyanine; and the first component is a carbocyanine dye and the second component is naphthalocyanine.
In other preferred embodiments, the invention relates to particles comprising an energy donor as a first component and a fluorescent dye as a second component positioned in a particle at an energy exchanging distance from one another, the two components having a Stokes shift o

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