Fluorescence is a physical phenomenon based upon the ability of some molecules to absorb and emit light. With some molecules, the absorption of light (photons) at specified wavelengths is followed by the emission of light from the molecule of a longer wavelength and at a lower energy state. Such emissions are called fluorescence and the emission lifetime is said to be the average period of time the molecule remains in an excited energy state before it emits light of the longer wavelength. Substances which release significant amounts of fluorescent light are termed "fluorophores" and are divisible into two broad classes: intrinsic fluorescent substances and extrinsic fluorescent substances. Intrinsic fluorophores comprise the naturally occuring biological molecules whose demonstrated ability to absorb exciting light and emit light of longer wavelengths is directly based on their internal structure and chemical formulation. Typical examples include proteins and polypeptides containing tryptophan, tyrosine, and phenylalanine. In addition, enzymatic cofactors such as NADH, FMN, FAD, and riboflavin are highly fluorescent. Extrinsic fluorophores, for the most part, do not occur in nature and have been developed for use as dyes to label proteins, immunoglobulins, lipids, and nucleic acids. This broad class includes fluorescein, rhodamine, and their isocyanates and isothiocyanate derivatives; dansyl chloride; naphthylamine sulfonic acids such as 1-anilino-8-naphthalene sulfonic acid ("ANS") and 2-p-toluidinylnaphthalene-6-sulfonic acid ("TNS") and their derivatives; acridine orange; proflavin; ethidium bromide; quinacrine chloride; and the like.
Substances able to fluoresce share and display a number of common characteristics: fluorophores display the ability to absorb light at one wavelength or frequency, reach an excited energy state, and subsequently emit light at another light frequency and energy level. The excitation spectrum and fluorescence emission spectrum are individual for each fluorophore and are often graphically represented as two separate curves which are slightly overlapping. The absorption and emission spectra for quinine bisulfate is depicted in FIG. 1 and is representative of fluorescent substances in general. All fluorophores demonstrate the Stokes' Shift--that is, the emitted light is always at a longer wavelength (and at a lower energy level) relative to the wavelength (and the energy level) of the exciting light absorbed by the substance. Moreover, the same fluorescence emission spectrum is generally observed irrespective of the wavelength of the exciting light; accordingly, the wavelength and energy of the exciting light may be varied within limits, but the light emitted by the fluorophore will provide the same emission spectrum. Finally, fluorescence may be measured as the quantum yield of light emitted; the fluorescence quantum yield is the ratio of the number of photons emitted in comparison to the number of photons absorbed. The quantum yield of photons and the timed duration over which that quantity of emitted light is detectable may be modified by a variety of factors. For more detailed information regarding each of these characteristics the following references are recommended: Lakowicz, J. R., Principles Of Fluorescence Spectroscopy, Plenum Press, New York, 1983; Freifelder, D., Physical Biochemistry, Second Edition, W. H. Freeman and Company, New York, 1982.
Analytical methods utilizing extrinsic fluorophores have had to conform to several specific requirements in order to be useful: (1) The extrinsic fluorophore must be capable of being tightly bound at a specific location or reactive to a unique chemical entity. (2) Its fluorescence must be sensitive to those changes in the environmental test conditions or system indicative of a chemical change. (3) The extrinsic fluorophore should not directly affect the features or properties of the molecule being investigated. Alternatively, however, the user has the option to use extrinsic fluorophores that react with an analyte in either a reversible or irreversible manner. If used irreversibly, fresh fluorophore must be added to the test sample each time an analysis is performed. In practical effect, this has required the investigator to adapt one of two functional approaches: to utilize fluorophores which themselves demonstrate the capacity to specifically bind to a preselected ligand or analyte of interest; or to alteratively chemically combine a non-specific fluorophore with another composition which has the requisite specific binding capacity to form a conjugate molecule, the binding specificity of the conjugate being provided by the other compound while the light emitting capability is provided by the fluorophore. Each approach is exemplified by the presently known qualitative and quantitative assay methods now well established in the art.
For example, the fusion of an extrinsic fluorophore with a specific antibody has been employed in two applications. First, by the use of such fluorescent labelled specific antibody for the study of specific macromolecules or cells in tissue sections. Specific cells or tissues are combined with a conjugate comprising the fluorophore and the antibody which is then applied over a section of prepared tissue; the attachment of the fluorescent labelled antibody identifies the existence of a specific macromolecule within the tissue sections [Coons, A. H., Int. Rev. Cytol. 5:1 (1956); Coons, A. H., "Fluorescent Antibody Methods," in General Cytochemical Methods (Danielli, J. F., Editor) Academic Press, New York, 1958, pp 399-422; Saint-Marie, G., J. Histochem. 10:250 (1962)].
A second example is the use of extrinsic fluorophores in immunoassays:: The fluorescent substance is again utilized as an identifying label with an immunogen whose presence in homogeneous and/or heterogeneous assays identifies the presence of a specific partner. Typically these include antibody-antigen reactions in competitive and non-competitive protocols [White, R. G., "Fluorescent Antibody Techniques," in Immunological Methods, (Ackroyd, J. F., Editor), Blackwell, Oxford, 1964; Nairn, R. C., Fluorescent Protein Tracing, Livingstone, Edinborough; Goldstein, J. Exp. Med. 114:89 (1961); Humphrey and White, Immunology For Students Of Medicine, Blackwell Scientific Publications, Oxford, England, 1966, pp 226-228].
Examples of using the fluorophore alone include the determination of the heme-binding site in hemoglobin. Hemoglobin is a complex of a small prosthetic group with the protein, apohemoglobin. The extrinsic fluor 1-anilino-8-naphthalene sulfonate (hereinafter "ANS") fluoresces when added to solutions of apohemoglobin but does not fluoresce with hemoglobin alone. The addition of heme to the apohemoglobin-ANS complex eliminates fluorescence by displacement of the ANS; accordingly, the site of attachment for the ANS and for heme must be the same. The timing as well as the location of such binding sites is identified by the fluorescence or elimination of fluorescence provided by ANS.
Similarly, the detection of a conformational change in an enzyme when the substrate becomes bound is detectable by the use of a fluorophore. 2-p-toluidylnaphthalene-6-sulfonate (hereinafter "TNS") fluoresces only if bound to another molecule; TNS fluoresces when added to the enzyme, alphachymotrypsin. The addition of a specific substrate for this enzyme decreases the fluorescence. Accordingly, fluorescence may be used as the means to determine both the degree of binding and the specific catalytic site for the enzyme with respect to the affinity and the location of the enzymatic reactions.
Fluorescence microscopy is another technique which has been utilized with fluorophores which have specific binding capacity and which can be transported and localized intracellularly. For example, acridine orange binds specifically to nucleic acids and fluoresces green or orange if bound to either DNA or RNA respectively. This technique has been used with eukaryotes to observe nucleic acids and chromosomes and to detect RNA in the nucleus; it has also been used with prokaryotes to localize and identify DNA.
By its nature and applications, fluorescence and fluorescent detection methods are recognized as being completely different and distinguishable from light energy absorbance and absorption spectroscopy. When light energy waves encounter a molecule, the light energy can either be scattered or absorbed. If the light energy is absorbed, the molecule is said to be in an excited state and is often termed a "chromophore" or an "absorber." Molecules which absorb light and do not fluoresce usually convert the light energy into heat or kinetic energy unlike fluorescent molecules which re-emit the light at lower energy levels. The ability to internally convert the absorbed light energy rather than emit it as light of another wavelength is a primary difference between absorbers and fluorophores.
Molecules which absorb light energy do so at individual wavelengths and are characterized by a distinctive molar absorption (extinction) coefficient at that wavelength. Chemical analyses utilizing absorption spectroscopy using visible and ultraviolet light wavelengths in combination with the absorption (extinction) coefficient allow for the determination of concentration for specific compositions, for the assay of chemical reactions, and for the identification of individual compounds by spectral measurement. The most common use of absorbance measurement is to determine concentration which is calculated in accordance with Beer's law; accordingly, at a single absorbance wavelength, the greater the quantity of the composition which absorbs light at the single wavelength, the greater the optical density for the sample. In this way, the total quantity of light absorbed is directly correlated with the quantity of the composition in the sample.
Another application lies in those chemical reactions in which one of the reactants changes its absorbance characteristics during the course of the reaction; a common example from enzymology is the use of an enzyme to convert a substrate into a product or products, in which the substrate and/or product absorbs light at a given wavelength. Accordingly, the more of the reaction product that is formed, the greater the change in the quantity of light absorbed. The optical density change over time thus provides a quantitative measure of the activity for that enzyme.
In addition to these, some very sophisticated model systems employing fluorescence have been developed. These include: quenching of fluorescence; and energy transfer system utilizing fluorescent light energy. Basic principles of both these systems are well described within Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York and London, 1984, pages 257-339.
Fluorescence quenching refers to any process which decreases the fluorescence intensity of a given substance and can occur via a variety of different mechanisms such as excited state reactions, energy transfer, complex formation, and collisional quenching. Quenching can be dynamic (or collisional) in nature which results from collisional encounters between a fluorophore and a quencher molecule; alternatively, quenching can be static in nature and result from the formation of complexes between the fluorophore and the quencher. In comparison, fluorescence energy transfer is a mechanism of action in which a transfer of the excited state energy is made from a donor molecule to an acceptor entity. This kind of transfer occurs without the appearance of a photon from the donor and is thus non-radiative in nature; and is deemed to be primarily the result of dipole-dipole interactions between the donor and acceptor compositions. The rate of non-radiative energy transfer depends upon the extent of overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the relative orientation of the donor and acceptor dipoles, and the spatial distance between the donor and acceptor molecules. It is this latter limitation and dependence upon spatial distance between the acceptor and donor molecules which has led to the now common use of energy transfer as a means for measuring distances between a donor and acceptor in solution. Detailed investigations of fluorescence quenching techniques based on energy transfer mechanisms of action have been reported [Anufrieva, E. V. and Y. Y. Gotlib, Adv. Polym. Sci. 40:1 (1981); Slomkowski, S. and M. A. Winnik, Macromolecules 19:500 (1986)].
In the traditional and established viewpoint of practitioners in this art, each of the conventionally known fluorescence phenomenon and fluorescent mechanisms of action have been deemed to be individually distinct and separate from one another. The underlying principles established for each physical phenomenon are unique and distinguishable from even closely related light energy systems. Insofar as is presently known, therefore, there has been no method or technique which has physically joined a fluorophore and an absorber together as a conjugate composition and then employed the conjugate composition in an energy transfer mechanism of action for the quantitative and/or qualitative detection of an analyte.