Dipyrrometheneboron difluoride labeled flourescent microparticles

#The invention relates to microparticles labeled with fluorescent dyes. In particular, the invention describes polymer microparticles labeled with dipyrrometheneboron difluoride derivative compounds, optionally including additional dyes and/or bioreactive materials, for use in standardization of fluorescence-based instrumentation, as biological tracers and in the detection and analysis of biomolecules.

#Polymer microparticles labeled with fluorescent dyes (fluorescent microparticles) have found use in a wide variety of applications. Microparticles are generally considered to be spherical or irregular in shape, and to be less than about 50 micrometers in diameter. They may be prepared by several practical methods from a variety of polymerizable monomers, including styrenes, acrylates and unsaturated chlorides, esters, acetates, amides and alcohols. For example, Rembaum (U.S. Pat. No. 4,326,008 (1982)) discloses fluorescent microspheres obtained by copolymerizing an acrylic monomer containing a covalent bonding group such as hydroxyl, amine, or carboxyl with a fluorescent co-monomer such as dansyl allyl amine. Fluorescent microparticles are most commonly used in applications that can benefit from use of monodisperse, chemically inert, biocompatible particles that radiate detectable fluorescence and that can bind to a particular substance in the environment.

#For example, fluorescent particles to which biological molecules have been attached have been used for immunoassays (U.S. Pat. No. 4,808,524 to Snyder, et al. (1989)), as labels for cell surface antigens (Jett, Keller, Martin, Nguyen, & Saunders, Ultrasensitive Molecular-Level Flow Cytometry, in FLOW CYTOMETRY AND SORTING, p. 381 (2nd ed., Wiley-Liss Inc., N.Y. 1990)), and as tracers to study cellular metabolic processes (Hook & Odeyale, Confocal Scanning Fluorescence Microscopy: A New Method for Phagocytosis Research, J. LEUKOCYTE BIOL. 45: 277 (1989)). The high surface area of microparticles provides an excellent matrix for attaching biological molecules while the fluorescent properties of these particles enable them to be detected with high sensitivity. They can be quantitated by their fluorescence either in aqueous suspension or when captured on membranes. Fluorescent microparticles can be visualized with a variety of imaging techniques, including ordinary light or fluorescence microscopy and laser scanning confocal microscopy. Three-dimensional imaging resolution techniques in confocal microscopy utilize knowledge of the microscope's point spread function (image of a point source) to place out-of-focus light in its proper perspective. Small, uniform fluorescent labeled polystyrene microspheres have been employed as point sources for these microscopes (Cartington, Fogarty, Lifshitz, & Fay, Three-Dimensional Imaging on Confocal and Wide-Field Microscopes, in HANDBOOK OF BIOLOGICAL CONFOCAL MICROSCOPY Ch. 14, p. 154 (Rev. ed. J. Pawley, Plenum Press, N.Y. 1990).

#FIG. 1: The relative fluorescence of polystyrene beads labeled with Compound 1 and coumarin 6. Spectra A) The emission spectrum of 1.09 micrometer polystyrene latex particles containing 50 mmol/gram of Compound 1 (see Table 1 below), excited at 490 nm (a preferred excitation wavelength). Spectra B) The emission spectrum of an identical concentration of identical latex particles containing 50 mmol/gram of coumarin 6, excited at 460 nm (excitation maximum).

#FIG. 2: A comparison between a dipyrrometheneboron difluoride dye and another fluorescent dye relative to the consistency of the spectral response for the dyes in solvent and incorporated internally in the microparticles. FIG. 2A shows the normalized emission spectra of Compound 1 in methanol solution (spectrum A) and incorporated into 30 nm polystyrene microparticles (spectrum B). FIG. 2B shows the normalized emission spectra of coumarin 6 in methanol solution (spectrum C) and incorporated into 30 nm polystyrene microparticles (spectrum D). Each set of spectra are plotted against the same x-axis scale, from 455 to 700 nm. Since the spectra in FIGS. 2A and 2B are normalized (i.e. adjusted to the same peak height) for ease of comparison, the peak height in FIGS. 2A and 2B is not indicative of relative fluorescence intensity for either compound in solvent or after incorporation into the microparticles. The spectrum of Compound 1 shifts very little upon incorporation into the microparticle. In contrast, the very broad emission of coumarin 6 shifts in both emission wavelength and bandshape upon being incorporated into the microparticle, and becomes even broader.

#FIG. 3: The ability of multiple dipyrrometheneboron difluoride dyes to match the spectrum of a target material (Example 4). FIG. 3A shows the spectra of microparticles I and II, each labeled with a different dipyrrometheneboron difluoride dye (spectra A and B, respectively), in comparison with the spectrum of the target material: a bead surface-labeled with fluorescein isothiocyanate ("FITC bead", spectrum C). FIG. 3B shows the spectrum for microparticles (III) containing a mixture of dyes used for A and B (solid line), in comparison with the spectrum of the FITC bead target material (dashed line).

#The novel fluorescent microparticles are prepared using derivatives of dipyrrometheneboron difluoride that are incorporated internally in polymeric microparticles. Typically, the dipyrrometheneboron difluoride dyes are derived from the dyes described in U.S. Pat. No. 4,774,339 (incorporated by reference) modified by substitution on either of the pyrrole rings or the non-boron linking group between these rings. Appropriate selection of substituents gives dipyrrometheneboron difluoride derivatives that, when incorporated into polymer particles, give desired excitation and emission wavelengths. Microparticles labeled by incorporation of dipyrrometheneboron difluoride dyes inside the microparticles retain the advantageous spectral properties of the dyes, such as the high extinction coefficient of the dyes (typically greater than 25,000 cm.sup.-1 M.sup.-1, preferably greater than 50,000 cm.sup.-1 M.sup.-1, more preferably greater than 70,000 cm.sup.-1 M.sup.-1) and high quantum yield of the dyes (typically greater than 0.1, preferably greater than 0.2, more preferably greater than 0.5).

#In one aspect of the invention, dipyrrometheneboron difluoride derivatives used for the preparation of fluorescent polymer microparticles according to this invention generally have the structure: ##STR1## wherein R.sup.1 -R.sup.7, which may be the same or different, are hydrogen, halogen, alkyl, alkoxy, aryl, arylalkyl, cycloalkyl, acyl, formyl, or cyano. Typically, R.sup.1 -R.sup.7, which may be the same or different, are hydrogen, alkyl-, alkoxy-, aryl-, or halo-; and X is --CR.sup.4 .dbd., where R.sup.4 is hydrogen; or alkyl-, aryl-, or heteroaryl-substituted. More typically, R.sup.1 -R.sup.7 are independently a hydrogen, hydrocarbon, or heteroaryl substituent.

#Where microparticles with longer wavelength excitation and emission are desired, the dipyrrometheneboron difluoride derivatives used for the preparation of fluorescent polymer microparticles according to this invention generally have the structure described above, excepting that any or all of the substituents R.sup.1 -R.sup.7, but at least one of such substituents, is an unsaturated organic group, i.e. a substituted or unsubstituted aryl, alkenyl, or heteroaryl group, or adjacent substituents R.sup.2 and R.sup.3 and/or R.sup.5 and R.sup.6 in combination form 1 or 2 fused unsaturated ring substituents, where each fused ring is optionally and independently substituted or unsubstituted. Dipyrrometheneboron difluoride dyes that are triazaindacene rather than diazaindacene dyes (where X is nitrogen) are equivalent to some diazaindacene dyes substituted with an unsaturated organic group in achieving desired red-shifting of microparticle emission spectra. Multiple unsaturated organic substituents, in combinations of R.sup.1 -R.sup.7 may be the same or different. Generally, the remaining substituents are hydrogen, halogen, alkyl, alkoxy, arylalkyl, cycloalkyl, acyl, or formyl. Typically, remaining substituents are hydrogen or alkyl.