Fluorescence energy transfer and intramolecular energy transfer in particles using novel compounds

FIELD OF THE INVENTION
This invention relates generally to the synthesis of novel dyes and labels and methods for the detection or visualization of analytes and more specifically to fluorescent latex particles which incorporate the novel fluorescent dyes and utilize, in certain aspects, fluorescence energy transfer and intramolecular energy transfer, for the detection of analytes in immunoassays or in nucleic acid assays.
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 fluorescence is generated by excitation of the dye at the appropriate wavelength. 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 a background signal. When 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 fluorescent particles. 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.
Many novel phthalocyanine derivatives and hybrid phalocyanine derivatives are disclosed and claimed. In one embodiment microparticles having at least one hybrid phthalocyanine derivative, said derivative(s) having (1) at least one donor subunit with a desired excitation peak; and (2) at least one acceptor subunit with a desired emission peak, wherein said derivative(s) is/are capable of intramolecular energy transfer from said donor subunit to said acceptor subunit are disclosed. Such derivatives also may contain an electron transfer subunit. Axial ligands may be covalently bound to the metals contained in the hybrid phthalocyanine derivatives. Numerous compounds capable of intramolecular energy transfer as well as compounds for fluorescence energy transfer are claimed.

DESCRIPTION OF THE DRAWING
FIG. 1 depicts the structures of phthalocyanine, naphthalocyanine and anthranylocyanine.
FIG. 2 depicts the structures of silicon phthalocyanine, silicon naphthalocyanine and silicon anthranylocyanine.
FIG. 3 depicts the spectra of silicon phthalocyanine dihydroxide and the spectra of silicon 2,3-naphthalocyanine dihydroxide.
FIG. 4 depicts the general structure of ethenyl-substituted dipyrrometheneboron difluoro dyes.
FIG. 5 depicts the attenuation of the background signal as a function of increasing wavelength. The data was measured using a device as described in Applicant's allowed Ser. No. 07/887,526 filed May 21, 1992 entitled "Diagnostic Devices and Apparatus for the Controlled Movements of Reagents Without Membranes," now U.S. Pat. No. 5,458,852 which is hereby fully incorporated herein.
FIG. 6 depicts naphthalocyanine derivatives which emit in the near infrared.
FIG. 7 depicts general structures of fluorescent energy transfer naphthalocyanine compounds.
FIG. 8 depicts the absorbance spectrum of human serum between 200 nm and 1000 nm.
FIG. 9 depicts the structure of a novel hybrid phthalocyanine derivative, Silicon �di(1,6-diphenylnaphthalocyanine)! diphthalo-cyanine bis(dimethylhexylvinylsilyloxide).

DETAILED DESCRIPTION
This invention describes novel fluorescent particles and novel fluorescent molecules and diagnostic methods for their use. Developing a method for the visualization of a cellular component or a cell or for an assay which utilizes a fluorescent dye and which quantifies an analyte in a sample requires the use of a fluorometer. The fluorescent label, the sample and the instrument must be compatible with each other to achieve an accurate measurement. Several criteria for a fluorescent label as it relates to the sample and instrument are described below. First, the absorption or excitation and emission wavelengths of the dye should not correspond to those of the specimen or sample. Second, the Stokes shift of the dye should be as large as possible to minimize the measurement of background from the excitation wavelength. Third, the dye must be compatible with the phase of the visualization or the fluid phase of the assay; that is, the dye must be water soluble or water insoluble depending on the visualization or assay format. Fourth, the dye should be as bright as is necessary to achieve the desired sensitivity. Brightness is the product of the extinction coefficient and the quantum yield of the dye. Fifth, the instrument used to detect the fluorescent signal is generally designed around the specifications of the dye and the specimen or sample being visualized or assayed.
These points will be discussed in more detail and illustrate some of the difficulties in developing a fluorescent visualization technique or an assay using fluorescent dyes. One is limited either to dyes which have been synthesized or ones which must be synthesized in order to meet the above criteria. Those skilled in the art will appreciate that the design and synthesis of dye molecules which have a very broad range of excitation and emission wavelengths is very tedious and generally, only a very limited range of excitation and emission wavelengths can be planned for a specific molecule. The teachings of this invention allow one to prepare fluorescent labels which can be tuned to many excitation and emission wavelengths allowing for large Stokes shifts. Thus, designing a dye system with the specifications of the sample or specimen and the instrument is possible rather than designing the instrument around the specifications of the dye. Tuning the dye system to accommodate the characteristics of the sample and the instrument results in a much greater chance of success of the visualization process or the assay.
The excitation and emission wavelengths of the dye should not correspond to those of the sample being assayed or visualized, otherwise the sample can interfere with the measurement of the fluorescent signal. When absorption or emission wavelengths of the sample do correspond to those of the dye, in practice, one dilutes, for example, a serum or blood sample so that the interference by the sample is reduced or the interfering sample is washed away from the detection area. Indeed, currently on the market, no fluorescent assay system exists for the measurement of analytes in neat biological fluids, particularly blood or serum. One reason for the lack of fluorescent assay systems which detect analytes in neat samples is that no good fluorescent dye exists which meets all the criteria listed above, particularly for measuring fluorescence in biological samples. When the sample absorbs significantly at the excitation wavelength the amount of light which excites the sample is thus affected by the variation in the sample characteristics. For example, serum, plasma, or blood from different individuals will be different in their relative absorptivities, which will translate into different intensities of excitation light used to excite the fluorescent label. The fluorescence emission of the dye is directly proportional to the intensity of the incident light, such that when the sample absorbs a portion of the incident light, the intensity of the fluorescent signal will vary accordingly. This results in measuring an incorrect or effected fluorescence emission. In addition, the emission wavelength of the dye should not correlate with the emission or absorbance of the sample because the sample will increase the measured fluorescence of the dye or the sample will absorb all or a portion of the dye fluorescence and also result in an incorrect or effected fluorescence emission. These problems are avoided when the sample is invisible to the excitation and emission wavelengths.
FIG. 8 shows the spectrum between 200 nm and 1000 nm of human serum. Wavelengths above 600 nm absorb considerably less than those between 200 nm and 600 nm. Thus, both the absorption of the incident light and the effect on the fluorescence of a dye are minimal when exciting above 600 nm. Preferred excitation wavelengths for biological fluids, including urine, blood, serum or plasma is 600 nm or greater. Particularly preferred excitation wavelengths above 600 nm are those which correspond to the maximum light output of laser diodes and light emitting diodes. Preferred emission wavelengths are those above 600 nm. The intrinsic sample fluorescence can cause a high background signal if the emission wavelength of the dye and the sample are overlapping. In addition, the scattered light of the excitation source can also contribute to the background signal. The contribution of the scattered light to the background can be seen, for example, in FIG. 5. In general, the magnitude of the scatter is inversely proportional to the fourth power of the measured wavelength. This teaches that desired emission wavelengths are in the near-infrared or in the infrared region of the spectrum. The inventive teachings described herein provide for dyes and dye systems which excite above 600 nm and which emit above 650 nm and more preferred, above 730 nm.
The Stokes shift of the dye should be as large as possible to minimize the measurement of background from the excitation source so that the signal-to-background ratio at the limit of sensitivity is maximized. A large Stokes shift, however, will only maximize the efficiency of the fluorescence measurement and may not always result in an accurate fluorescence measurement. For example, table 3 shows data from several dye systems which were excited between 420 nm and 670 nm in either buffer or undiluted human serum. The fluorescence intensity of the first dye system (from line 1, table 1), when excited at 475 nm in serum, is only 7.6% of the intensity in buffer even though the Stokes shift is 205 nm. The second dye system (from line 4, table 1), excited at 420 nm, is 28% of the intensity in buffer with a 260 nm Stokes shift. The third and fourth dye systems (from line 60 and line 59, table 1), excited at 670 nm and 650 nm and with 110 nm and 130 nm Stokes shifts, respectively, have fluorescence intensities which are comparable in buffer and in serum. The fifth dye system, which is a hybrid phthalocyanine derivative (from line 1, table 2), has comparable fluorescence intensities in buffer and serum when excited at 646 nm with a Stokes shift of 114 nm. The data show that the fluorescence intensity is greatly affected when the excitation wavelength is within the range of the absorbance of the sample in which the measurement is made. The data also show that the magnitude of the Stokes shift does not have an influence on the accuracy of the measurement. These data are representative of other dyes and dye systems which are excited at a wavelength where the sample absorbs. The effect of the decreased fluorescence emission is not a result of the emission wavelength (that is, 680 nm or 780 nm) because both serum and buffer solution absorb minimally at 680 nm and 780 nm. One skilled in the art can appreciate, that with the inventive teachings described herein, the wavelengths for excitation and emission of a dye system should be a function more of the absorption and emission characteristics of the sample rather than selecting only a dye system with a large Stokes shift.
The availability of dyes with Stokes shifts greater than 100 nm is greatly limited, particularly when the excitation wavelength is greater than 600 nm. To further limit the usefulness of available dyes, the solubility of the dyes in aqueous samples can be a problem because most dyes with large Stokes shifts are water insoluble.
The problem of a dye possessing a small Stokes shift is usually overcome in the engineering of the fluorometer by the use of monochromators or expensive optics which filter out the light from the excitation source. However, to overcome the loss in light intensity due to the filters, for example, one requires the use of high powered light sources. These light sources produce heat which must be dissipated in an instrument by using heat sinks or fans. The complexity of the fluorescence measuring device, both from an optical and a mechanical perspective, is thus greatly affected by the inadequacies of the dye system. With the advent of near-patient testing in hospitals and emergency departments, instruments which measure fluorescence in immunoassays will be required to be portable and uncomplicated to the technician. Thus, the future state of the art for the manufacture of, for example, fluorometers which are employed for immunoassays will be required to change to simple and portable instruments. The high powered light sources and expensive optics currently incorporated into fluorometers will not meet the requirements for small, portable instruments. The inventive features of the instant invention teach that fluorescent labels can be prepared with large Stokes shifts and be tuned to wavelengths both of which are compatible with excitation sources and emission detectors and which are compatible with the absorption and emission of the sample, for example, blood, serum, plasma, urine, ground water, and the like. The excitation and emission wavelengths of the novel fluorescent particles can generally be varied independently of each other.
The dye must be compatible with the fluid phase of the assay, or in other words, the dye must be water soluble or water insoluble depending on the visualization or assay format. Many fluorescent dyes are water insoluble or poorly water soluble and these dyes are not easily used for labelling molecules, proteins, nucleic acids or cells. One skilled in the art will recognize that water insoluble dyes can be incorporated into latex particles as described in U.S. Pat. Nos. 4,326,008, 4,609,689 and 5,154,887, which are hereby incorporated by reference. Thus, water insoluble dyes can be made useful by incorporation into latex particles for visualization in a variety of assay formats.
The dye should be as bright as is necessary to achieve the desired sensitivity. If one knows the extinction coefficient and the quantum yield of the dye and the concentration of the target to be measured, it can be estimated whether the dye is bright enough to achieve the desired sensitivity. Incorporation of dyes into latex particles or the utilization of an enzyme which catalyzes the production of a fluorescent substrate are examples of techniques which one skilled in the art uses as amplification systems.
The instrument used to detect the fluorescent signal is generally designed around the specifications of the dye and the specimen or sample being visualized or assayed because of the limited numbers of dyes which can be successfully used. As discussed above, the components of the instrument are selected for a particular dye system since a useful instrument must be highly tuned to eliminate the light from the excitation source.
Each of the conditions described above, taken together, greatly narrows the development of dye systems which can be employed for measuring sub-picomolar concentrations of analytes, particularly in biological fluids. The limitations also impose restrictions on the design of an instrument to measure the fluorescence. The novel teachings of the instant invention allow the design, synthesis and tuning of dye systems to match, generally, nearly any instrument design.
Several inventive teachings are described for tuning excitation and emission wavelengths of dyes so that the excitation and emission are compatible with the sample matrix in which the fluorescence is measured and the instrument for quantifying the fluorescence. One teaching is to either incorporate or adsorb at least two dyes into or onto particles, which, as a pair, exhibit fluorescence energy transfer. The particles which can be used are those which absorb dyes on the surface or inside the particle. Another teaching is to incorporate dyes which are covalently attached to each other and which also exhibit fluorescence energy transfer both in solution and in particles. Yet another teaching is to incorporate hybrids of phthalocyanines, naphthalocyanines, anthranylocyanines and derivatives of these classes of compounds.
The selection of dye pairs for incorporation into particles is based on their ability to exhibit energy transfer (singlet-singlet energy transfer) at the appropriate excitation wavelength of the donor dye and the emission of the acceptor. Fluorescence energy transfer of two molecules is well known to those skilled in the art and the rate of energy transfer is described by Forster in Ann. Physik. (1948) 2,55-75. Fluorescence energy transfer has been used as a spectroscopic ruler to predict proximity relationships in proteins, RNA and peptides (Annual Review of Biochemistry (1978), 47, 819-846) and also to probe geometrical details in particles (Physical Review Letters (1988) 61, 641-644). U.S. Pat. No. 5,326,692 describes fluorescent particles with controllable enhanced Stokes shifts. U.S. Pat. Nos. 4,542,104 and 4,666,862 describe fluorescence energy transfer in phycobiliproteins. These dye complexes are described for use as labels in immunoassays; however, the limited use of phycobiliproteins and the expense of these natural protein complexes make them undesirable for use on a commercial scale. Unsymmetrical or hybrid phthalocyanines have been described, for example, in J. Am. Chem. Soc. 1990, 112, 9640-9641, Chemistry Letters 1992, 2031-2034 and Inorg. Chem. 1994, 33, 1735-1740 but the inventive teachings described herein expand the potential compounds which can be synthesized for use in immunodiagnostics to achieve adequate fluorescence intensities and desired excitation and emission characteristics. The inventive teachings described herein also teach that the ratio of the various diiminoisoindiline or dicarbonitrile precursors and their substitution by electron donating or electron withdrawing groups in the synthesis of the hybrid phthalocyanines, naphthalocyanines and anthranylocyanines will affect the absorption spectrum and the excitation and emission wavelengths of the compounds.
In one aspect, the novel fluorescent particles of this invention are composed of at least two dyes which are positioned in the interior or on the exterior of particles at an energy exchanging distance. One skilled in the art will recognize that various particles can be utilized, such as latex, silica, alumina, liposomes, various colloids and the like. Particularly preferred particles are latex particles. The selection of the dye molecules for incorporation into the particles should be related to the specific use of the particles, the sample to be analyzed and the instrument for measuring the fluorescence. For example, when developing an assay for an analyte in a biological medium, such as serum or a cell extract, the intrinsic absorbance and fluorescence of the sample must be considered. Serum and cellular components absorb in the ultraviolet spectrum as well as in the visible spectrum up to around 600 nm and the intrinsic fluorescence can broadly approach 600 nm. In addition, samples which contain small particles, such as dirt particles in ground water, lipoproteins in serum or blood, cells and cellular particles and components will scatter the excitation light which results in a higher background signal. The ideal dye couple would include the donor dye which would be excited or absorb at above 600 nm and emit at a wavelength which the acceptor dye absorbs, and the acceptor dye should emit at a wavelength above 600 nm. In the case of a single dye system, for example, with the use of hybrid phthalocyanine derivatives, the excitation and emission wavelengths should also be above 600 nm. The sample, for example, serum, then does not affect fluorescence of the acceptor dye because the sample poorly absorbs at the absorption of the donor dye and the acceptor dye emits at a wavelength where the sample does not fluoresce.
Fluorescent dye molecules incorporated into or onto particles will exhibit fluorescence quenching because of the close proximity of the dyes to each other and to the matrix of the particle. When loading dyes into or onto particles, one must optimize the concentration of dye as it relates to quenching. The dyes can be loaded successively or together. The degree of quenching can be quantified by measuring the fluorescence emission of a dilute suspension of particles (about 0.001% to 0.1% solids) in a buffer solution, in a buffered protein solution or in water and then also measuring the fluorescence of the same concentration of particles in solvent which liberates the dyes from the particles. The ratio of the fluorescence intensities (1-�fluorescence intensity of incorporated dyes divided by the intensity of liberated dyes! is the degree of quenching of the dyes in the particle. In practice, one incorporates dyes at various concentrations and measures the fluorescence intensities of the incorporated and liberated dyes to optimize the intensity of fluorescence of the particle while minimizing the quenching of fluorescence in the particle. In a situation where more than one acceptor dye is used to minimize fluorescence quenching and to maximize fluorescence intensity, one may use different acceptor dyes which have emission peaks which are within about 10 nanometers of one another. Another important consideration is the efficiency of the fluorescence energy transfer. In practice, if the energy transfer efficiency is not close to 100%, then one can observe the fluorescence of the donor dye. The resulting fluorescence of the donor dye can make the particles undesirable or even useless because the "effective Stokes shift" (that is, the shortest wavelength distance to a light source from the defined acceptor molecule emission wavelength in the fluorescence system) of the particles is now not the difference between the excitation and emission wavelengths of the donor and acceptor dyes, respectively, but rather the difference between the donor emission and the acceptor emission wavelengths. The emissions of the donor and acceptor wavelengths can overlap partially with each other when efficient energy transfer is not obtained and complicate the selection of filters for use in a fluorometer. The decrease in the energy transfer efficiency can also be directly related to a decrease in the emission of the acceptor dye, resulting in a particle which may not be as bright as a particle with efficient energy transfer. In addition, under conditions of inefficient energy transfer, slight changes in the sample or in solution conditions, for example, pH, ionic strength and the like, may affect the magnitude of energy transfer efficiency and thereby may affect the intensity of the fluorescent signal.
In selecting dye pairs for fluorescence energy transfer one begins by studying the overlap of the donor emission and acceptor excitation wavelengths. The dyes are positioned in the particle at an energy exchanging distance from one another which allows singlet-singlet energy transfer. Although a particular pair of dyes has acceptable overlapping excitation and emission wavelengths (for example, see Proc. Natl. Acad. Sci. USA 1969, 63, 23-30), they may not exhibit fluorescence energy transfer in particles or they may have suboptimal (less than 80%) efficiency of energy transfer. The process to determine whether 2 or more dyes will exhibit efficient energy transfer is through experimentation after the appropriate spectral overlap criteria are met. The efficiency of fluorescence energy transfer is determined by measuring the fluorescence intensity of the donor dye alone in particles and also measuring the fluorescence emission of the particles which have incorporated 2 or more dyes (that is, the fluorescent energy transfer particle) at the emission wavelength of the donor dye, both sets of particles having the same concentrations of donor dye and particles. The measured fluorescence at the donor dye emission wavelength of the fluorescent energy transfer particles divided by the fluorescence of the donor dye particles is the efficiency of fluorescence energy transfer. Ideally, in practice, the emission of the donor dye should be undetectable or only slightly detectable so that the effective Stokes shift is not reduced because of the donor dye emission. Preferred fluorescence energy transfer efficiencies are 80% or greater and particularly preferred fluorescence energy transfer efficiencies are 90% or greater.
The inventive teachings described herein provide for particles with reduced quenching and improved fluorescence intensities. A large majority of fluorescent molecules have aromatic character, that is, they possess 4n+2 pi electrons. The resultant aromatic character promotes stacking of the molecules, especially of water insoluble molecules in aqueous solutions or in particles in aqueous solution, which in turn promotes fluorescence quenching. The novel fluorescent particles described in the instant invention are incorporated with dyes which, through steric interference of the dye molecules, have a minimized propensity to stack in the particles. In another aspect of this invention, fluorescence quenching of dye molecules in particles is minimized by employing different dyes with approximately the same excitation and emission wavelengths. That is, the wavelength maximum for excitation and/or emission of the different dyes are within about 10 nm of each other so that there is substantial overlap of the peaks. One skilled in the art can appreciate that the width of excitation and emission spectra of various dyes can vary. The principle here is that different dyes will not stack in an organized orientation with each other to the same degree as dyes which are the same. An analogy to this stacking principle is the depression of the melting point of a pure compound by an impurity. It is well known to physical chemists that an impurity in a solid compound lowers its melting point because the impurity disrupts the formation of the crystal lattice of the pure compound. Incorporating dyes into or onto particles using organic solvents and then removing the solvent causes the dye to precipitate or crystallize in the particle. The disruption of the crystalline lattice of dye molecules in particles will alter the stacking of the molecules and thereby reduce quenching. Thus, incorporation of dissimilar dye molecules with similar excitation and emission spectra improves fluorescence intensities of the particles by decreasing the quenching interactions of the molecules.
In another aspect of this invention, incorporation into particles of dissimilar dyes which exhibit fluorescence energy transfer in the particles may also disrupt the other's crystalline lattice formation. Thus, the fluorescence intensities of particles exhibiting fluorescence energy transfer will be improved as a result of decreasing quenching in the particle because the stacking of similar dyes in the particles is disrupted by the dissimilar dye.
In yet another aspect of this invention, the synthesis of phthalocyanine derivatives and hybrid phthalocyanine derivatives with axial ligands reduces the stacking of the aromatic ring system, thus minimizing the interactions between molecules and maximizing fluorescence intensities.
One skilled in the art can appreciate that more than one dye pair which exhibits fluorescence energy transfer can be incorporated into or onto particles resulting in a class of particles which fluoresce at different wavelengths. In addition, with the inventive teachings described herein, incorporation into or onto particles of 3 or more dyes, which together provide a cascade of energy transfer from the absorber to the intermediate donor to the acceptor (which fluoresces), can result in the production of particles with very long Stokes shifts and allows one to produce particles with nearly an unlimited variety of excitation and emission characteristics.
FIG. 1 shows preferred acceptor dyes which are phthalocyanines, naphthalocyanines and anthranylocyanines. FIG. 2 shows particularly preferred acceptor dyes which are derivatives of silicon phthalocyanines, naphthalocyanines and anthranylocyanines, where R is hydrogen or an alkylcarbon chain from 1-20 carbons, either saturated or unsaturated, having 0-10 heteroatoms (N,O,S), and having 0 or 1 siloxide groups. The best mode compounds are those in which R=
Si(CH.sub.3).sub.2 C.sub.6 F5
Si(C.sub.6 H.sub.13).sub.3
Si(CH.sub.3).sub.2 (CH.sub.2).sub.3 CN
Si(CH.sub.3).sub.2 (CH.sub.2).sub.10 COOCH.sub.3
Si(CH.sub.3).sub.2 CH.dbd.CH.sub.2
Si(CH.sub.3).sub.2 (CH.sub.2).sub.10 COOH
Si(CH.sub.3).sub.2 (CH.sub.2).sub.4 Cl; and
Si(CH3).sub.2 (CH.sub.2).sub.6 CH.dbd.CH.sub.2.
The parent compounds of the phthalocyanines and naphthalocyanines are preferred because their emission wavelengths are around 680 nm and 780 nm in latex particles, respectively. Also preferred parent compounds are the anthranylocyanines which have emissions around 850 to 900 nm. These three classes of parent compounds will collectively be called "phthalocyanine derivatives" and may or may not have an included metal and may or may not have axial ligands. The emission wavelengths for the phthalocyanine derivatives are particularly useful for quantifying fluorescence in biological samples and for minimizing the background scatter intensity. Those skilled in the art can appreciate that phthalocyanine derivatives can be synthesized, for example, by derivatization of the phenyl, naphthyl or anthranyl rings with various substitutes to yield different molecules but these variants, also are within the scope of the instant invention. Derivatives of tetraazaporphine are also within the scope of the instant invention. The derivatization of the aromatic structure can produce blue or red shifted excitation or emission wavelengths. The choice of the donor dye to excite the phthalocyanine derivative dyes is dependent on having a donor dye emission wavelength which corresponds to the appropriate range of absorbance wavelengths of the phthalocyanine derivative. FIG. 3 shows the absorbance spectra of the silicon dihydroxyphthalocyanine and silicon dihydroxynaphthalocyanine in dimethylformamide. A potential range of excitation of the these acceptor dyes by the donor dye is between approximately 550 nm and 670 nm and 600 nm and 760 nm, respectively. One skilled in the art will recognize that many dyes would be candidates for the donor dye because of the wide useful range of wavelengths which can excite the acceptor dyes. The choice of the acceptor dye should meet the criteria outlined above. Several examples are described which illustrate the versatility of this novel approach. Assume that an instrument is to be built with an excitation source which has a maximum intensity at 480 nm and a detector which has a good quantum efficiency at 600 to 700 nm. The donor dye should thus be capable of being excited at 480 nm and further assuming that a phthalocyanine derivative is the acceptor dye for emission at 680 nm, the donor should then emit in the range of 550 to 670 nm.
Preferred classes of dyes for this application are styryl, phenylbutadienyl and phenylhexatrienyl dyes. Styryl dyes are those of the following formula: ##STR1## and phenylbutadienyl dyes are of the formula: ##STR2## and phenylhexatrienyl dyes are of the formula: ##STR3## wherein R1, R2 and R3 can be the same or different and R1, R2 and R3 are H or alkylcarbon chains from 1-20 carbons, either saturated or unsaturated, and having 0-10 heteroatoms (N, O, S).
In general, these dye classes excite approximately between about 470 and 530 nm and emit approximately between 600 and 780 nm (see Molecular Probes Handbook of Fluorescent Probes and Research Chemicals by Richard P. Haugland, 1992-1994, p. 156). A particularly preferred styryl dye is the trans-4-�4-(dibutylamino)styryl!-1-methylpyridinium iodide (Aldrich Chemical Co.) which has its maximum absorbance at 486 nm in dimethylformamide and its emission at 600 nm. One skilled in the art will recognize that the substituents off the aniline nitrogen and the pyridium nitrogen of these classes of dyes can vary and that preferred substituents are those with hydrophobic groups to maintain water insolubility.
In another application of this novel technology, an instrument system is built which has a source of maximum intensity at 420 nm and a detector as described in the above example. The dye system here can include the phthalocyanine acceptor; however, a different donor must be employed. A preferred donor for this application is a meso-tetra-2-aminophenylporphine (Porphyrin Products, Inc., Logan Utah) which has a maximum absorbance for excitation at 418 nm in dimethylsulfoxide and an emission around 655 nm. This porphyrin will excite the phthalocyanine derivative in latex particles and the dye system will emit at 680 nm.
In a particularly preferred application, an instrument system is built to perform immunoassays in neat blood or serum or in various biological specimens. The excitation source is an LED or laser diode which has its maximum intensity around 650 nm to avoid absorption of the light by the blood or serum sample. The detector has good quantum efficiency at 700 to 800 nm so a preferred acceptor dye is a naphthalocyanine derivative which has an emission at approximately 780 nm, an emission wavelength which is generally not in common with blood or serum samples or biological specimens. A donor dye for the naphthalocyanine acceptor should absorb at around 650 nm to coincide with the source and emit between approximately 660 nm and 760 nm. Preferred classes of dyes for this donor application are the carbocyanine dyes and the ethenyl-substituted dipyrrometheneboron difluoro dyes, as described in U.S. Pat. Nos. 5,187,288, 5,248,782 and 5,274,113.
In yet another particularly preferred application, for immunoassays in neat blood or serum, the excitation source is around 790 nm and the emission wavelength is around 900 nm. A preferred dye for a single dye system is a silicon 1,6-octaethoxynaphthalocyanine bis(dimethylhexylvinylsilyloxide) which is excited at 790 nm and emits at about 900 nm.
Preferred dyes for use as donor dyes for naphthalocyanines and naphthalocyanine derivatives are, carbocyanines and ethenyl-substituted dipyrrometheneboron difluoro dyes, as described in U.S. Pat. Nos. 5,187,288, 5,248,782 and 5,274,113 which have excitation wavelengths up to 790 nm and emission wavelengths between about 670 nm and 800 nm.
Preferred carbocyanine dyes, which generally excite between 500 and 750 nm (see Molecular Probes Handbook) are of the general formula: ##STR4## wherein n is 1 or 2; or 3; wherein R1 and R2 are S, N, or O; and wherein R3 and R4 are H or alkylcarbon chains of from 1-20 carbons, either saturated or unsaturated and having 0-10 heteroatoms (N, O, S).
Also preferred carbocyanine dyes are also of the general formula: ##STR5## wherein n is 1 or 2; or 3; wherein R1-R6 are H or alkylcarbon chains of from 1-20 carbons, either saturated or unsaturated and having 0-10 heteroatoms (N, O, S).
Preferred donor dyes are also the ethenyl-substituted dipyrrometheneboron difluoro dyes, which generally excite above 500 nm (see Molecular Probes Handbook) and are of the general formula as depicted in FIG. 4, wherein R1-R7 include substituents as described in U.S. Pat. Nos. 5,187,288, 5,248,782 and 5,274,113.
Particularly preferred donor dyes are 1,1'-dihexyl-3,3,3',3'-tetramethylindocarbocyanine iodide, 1,1'-diethyl-3,3,3',3'-tetramethylindodicarbocyanine iodide and (E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a, 4a-diazo-5-indacene (from Molecular Probes Inc., Eugene, Oreg.) which have absorption maximums of 642 nm, and 645 nm and 650 nm and emission maximums of 674 nm and 665 nm, and 670 nm, respectively, in dimethylformamide. Particles incorporated with these particularly preferred dyes and a naphthalocyanine derivative will excite with a 650 nm source and emit at approximately between 780 nm and 870 nm. One skilled in the art will recognize that the excitation and emission spectra for any particular dye has a Gaussian form and therefore the excitation source does not need to correspond exactly to the excitation maximum of the donor dye in order to obtain an intense fluorescent signal. Likewise, the donor emission does not have to coincide with the highest absorption of the acceptor dye in order to achieve efficient energy transfer. One skilled in the art will also recognize that the substituents at and on the 1 and 3 positions of the carbocyanines and the substituents at the R1 and R7 positions of the dipyrrometheneboron difluoro dyes, and the conjugation between the ring structures can vary and these variations are also useful in tuning fluorescence spectra of the particles.
Also preferred emission wavelengths of fluorescent particles range from about 800 nm to 1000 nm. This near infra-red region is important because the scattering component of the light decreases substantially, thus lowering the background of the fluorescent measurement. In addition, biological samples do not absorb or fluoresce substantially in the 800 nm-1000 nm range. Particulate materials in the samples, for example, lipoproteins in serum, particles in ground water, cellular debris in biological samples and the like, can increase the background signal because of scattered light and the measurement of the scattered light is minimized in the 800-1000 nm range. For example, FIG. 5 illustrates the attenuation of the background signal as the wavelength of the measured light increases from 730 nm to 900 nm in an immunoassay device, as described in allowed application Ser. No. 07/887,526 now U.S. Pat. No. 5,458,352 (which is herein incorporated by reference), containing either neat human serum or no serum. This figure shows that the background signal decreases by a factor of 5 when measuring at 900 nm as compared to 790 nm when the illumination source is a 1 milli watt ("mW") 670 nm laser diode. In addition, excitation of neat serum at 670 nm does not result in a significant measurable fluorescence between 730 nm and 900 nm. Thus, for example, the signal to background ratio of the measurement of fluorescence of a dye which emits at around 900 nm as compared to a dye emitting at around 790 nm would be improved by a factor of 5. The signal to background ratio improves by a factor of about 30 when measuring emission at 780 nm as compared to 730 nm (see FIG. 5). Maximizing the signal to background ratio, in general, is commonly sought in analytical chemistry because the sensitivity of the measurement is improved. Preferred dyes, for example as described in J. Chem. Soc. Perkin Trans. 1, (1988), 2453-2458, which emit above 780 nm include derivatives of the naphthalocyanine and anthranylocyanine classes (FIG. 1) and the naphthalocyanine class is characterized by the general formulae, as depicted in FIG. 6, where M is a metal such as Si, Ge, Al, Sn and Ti and the like, and where R is an axial ligand, alkyl or aryl with or without a silicon (preferred axial moieties are synthesized from alkyl or aryl silyl chlorides), and where X is an electron donating group or groups which can be the same or different, including, such as amino, hydroxyl, alkoxy, aryloxy, phenyl, alkyl and the like. The electron donating character of the X group or groups red-shifts the emission wavelength as compared to the general naphthalocyanine compounds (FIG. 1). For example, the compounds described in examples 26, 27 and 28 are illustrative of dyes which have emission wavelengths around 850 nm. These preferred dyes would yield an improved signal to background ratio as compared to dyes emitting at 780 nm (See FIG. 5). Electron withdrawing groups can also be utilized for the X groups, such as halogen, nitro, cyano, sulfate, carboxyl and carboxyalkyl and the like, which will blue shift the excitation or emission wavelengths. Preferred donor dyes for this class of near infra-red emitting dyes are those which have emission wavelengths which correlate to the absorbance characteristics of the acceptor dye. Preferred dyes for this application are the ethenyl-substituted dipyrrometheneboron difluoride dyes, as described in U. S. Pat. Nos. 5,187,288, 5,248,782 and 5,274,113.
Preferred molar ratios of donor to acceptor dyes in the latex particles generally range from about 20:1 to about 1:20 and particularly from about 1:1 to 6:1. The desired fluorescence intensity should be obtained through experimentation by incorporating various ratios of donor to acceptor dyes into the particles at various dye concentrations and measuring the fluorescence emission of the particles.
The geometrical orientation of the donor and acceptor dyes will affect the efficiency of energy transfer between them. The donor and acceptor dyes can be synthesized to form a compound of optimal geometry, which, in solution, exhibits efficient fluorescence energy transfer ("FET"). The optimized FET compound then may be incorporated into particles. Phthalocyanine derivatives can be utilized for this application for the acceptor moiety, where the phthalocyanine derivative can be substituted with electron donating or withdrawing groups (as described above) to accomodate the desired excitation and emission wavelength. For example, preferred naphthalocyanine compounds for this application are those as depicted in FIG. 7, where X is hydrogen or electron donating groups, such as amino, hydroxyl, alkoxy, aryloxy, phenyl, alkyl and the like and D is the donor dye covalently attached to the naphthalocyanine derivative at a distance which allows for energy transfer between the donor and acceptor. With the inventive teachings of the instant invention, one skilled in the art will recognize that all phthalocyanine derivatives can function as donor or acceptor molecules. For example, a silicon ortho octaethoxy(phthalocyanine) derivative will emit at approximately 750 nm to 780 nm, similar to a silicon naphthalocyanine derivative. Generally, the distances between donor and acceptor are about 5 angstroms to 60 angstroms, and preferably from 5 angstroms to 15 angstroms. In addition, each naphthalocyanine derivative can have 1-4 donor dyes attached, depending on the required application of the FET compound. Suitable donor dyes are those which emit in the absorbance range of the acceptor dye. Example 29 describes the synthesis of a fluorescein-silicon phthalocyanine FET compound. Table 1, item 56, shows the fluorescence characteristics of this compound in latex particles. One skilled in the art will appreciate that with the inventive teachings described herein, many FET compounds may be synthesized for many particular applications requiring specific excitation and emission wavelengths.
Another approach to developing particles which exhibit desired and predictable fluorescence properties in the high visible to near infrared spectrum is to synthesize unsymmetrical or hybrid phthalocyanines, naphthalocyanines or anthranylocyanines and their derivatives. The term "hybrid phthalocyanine derivatives" will herein refer to all classes of hybrid phthalocyanines, naphthalocyanines and anthranylocyanines and their derivatives, with or without metal and axial ligands, including tetraazaporphines and their derivatives. The novel hybrid molecules described herein appear to exhibit intramolecular energy transfer. The hybrid phthalocyanine derivatives can be synthesized from diiminoisoindoline or derivatives of diiminoisoindolines and incorporate a metal, for example, silicon, and elaboration with axial ligands or they can be synthesized from dicarbonitrile derivatives of benzene, naphthalene or anthracene compounds, respectively, for subsequent inclusion of various metals and elaboration with axial ligands. Hybrid molecules also comprised of derivatives of tetraazaporphines, as described in Inorg. Chem. (1994), 33, 1735-1740, are also within the scope of the hybrid phthalocyanine derivatives of the instant invention. A synthetic strategy for hybrid phthalocyanine derivatives with 2 different subunits is described, for example, in J. Am. Chem. Soc. (1990), 112, 9640-9641, Inorg. Chem. (1994),33, 1735-1740, Chem. Letters, (1992), 763-766, Chem. Letters, (1992), 1567-1570 and Chem. Letters, (1992), 2031-2034. These papers refer to the synthesis of hybrid molecules with zinc metal or without metal and without axial ligands. The versatility of the synthetic approach described herein as it applies to the inventive teachings of the instant invention is that the character of the diiminoisoindoline and its derivatives will dictate the excitation and emission characteristics of the molecule and, furthermore, elaboration with axial ligands will minimize quenching by decreasing stacking in the particles and maximize fluorescence intensity. Axial ligands on hybrid phthalocyanine derivatives are also beneficial on water soluble compounds because the axial ligands will minimize interaction of the hybrid molecule with, for example, proteins, antibodies and nucleic acids, which may or may not be covalently coupled to the hybrid molecule.
Novel hybrid phthalocyanine derivatives are described herein, which contain 3 or 4 different subunits, and allow for larger Stokes shifts. The principle is that excitation occurs with the subunit which has the highest energy or the lowest wavelength absorption and the emission occurs in the lowest energy subunit.
The desired excitation and emission wavelengths of the hybrid phthalocyanine derivative will determine the types of diiminoisoindoline derivative and dicarbonitrile derivative precursors which are used in the synthesis of the hybrid phthalocyanines. The desired excitation and emission wavelengths are generally dictated by the sample, the type of fluorescent measurement and the instrument. Various combinations of diiminoisoindoline derivative and dicarbonitrile derivative precursors may also combine to form a hybrid phthalocyanine derivative which may have a red shifted or blue shifted excitation and/or emission wavelength pattern. In general, electron donating substituents on the diiminoisoindoline or dicarbonitrile precursors, particularly situated at the ortho positions (that is, ortho to the tetraazaporphine structure as indicated in FIG. 6 for the X substituents) of the phthalocyanine structure, such as amino, hydroxyl, alkoxy, aryloxy, phenyl, alkyl and the like, will red shift the excitation and/or emission wavelengths. Conversely, generally, electron withdrawing substituents, also particularly at the ortho positions, such as halogen, nitro, cyano, sulfate, carboxyl and carboxyalkyl and the like, will blue shift the excitation or emission wavelengths. In addition, positions on the subunits other than the ortho positions can affect the excitation and emission characteristics of the hybrid phthalocyanine derivative. The choice of either diiminoisoindoline or dicarbonitrile precursors for the synthesis of the hybrid phthalocyanine derivatives is generally related to the desired presence or absence of metal and the type of metal in the hybrid molecule. For example, when using the diiminoisoindoline precursors in the synthesis, a silicon metal can be incorporated during the tetramerization reaction to form the phthalocyanine derivative structure. The silicon can be further modified to a silicon dihydroxy phthalocyanine derivative molecule so that axial ligands can be elaborated with, for example, various silyl chloride reagents. The importance of axial ligands in reducing quenching and maximizing fluorescence intensity is evident for both phthalocyanine/naphthalocyanine molecules and the hybrid phthalocyanine derivatives (see example 31). The axial ligands can also be useful for further elaboration of the molecules, for example, for attaching another fluorescent molecule, for attaching to a protein, polypeptide or nucleic acid or for changing the charge of the molecule using sulfate, carboxylic acid or amino substituents which can affect solubility of the molecule. When using the dicarbonitrile precursors, the phthalocyanine derivative is synthesized without metal, but various metals can subsequently be included, for example, Ge, Al, Sn, Ti and the like. These metals can also be elaborated with axial ligands, depending on the valence of the metal.
The fluorescence quenching character of the hybrid phthalocyanine derivatives are particularly preferred over the phthalocyanine derivatives. Example 32 is a typical example of comparison of the quenching characteristics in latex particles of silicon 2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide) and silicon �di(1,6-diphenylnaphthalocyanine)! diphthalocyanine bis (dimethylhexylvinylsilyloxide). The hybrid phthalocyanine derivative has essentially no quenching as compared to up to 50% quenching of the naphthalocyanine derivative for the various dye loading concentrations listed in the table. The fluorescence intensities of latex containing the hybrid phthalocyanine derivative are much greater than the phthalocyanine derivative. This illustrates the special properties of the hybrid phthalocyanine derivatives.
The tetramerization reactions of the diiminoisoindoline or dicarbonitrile precursors to form the hybrid phthalocyanine derivatives can be directed so that opposing subunits can be the same. This is accomplished, for example, with the use of bulky substituents on the precursors so that in the tetramerization reaction, like subunits with bulky substituents cannot be adjacent because of steric considerations. Bulky phenyl substituents have been used on dicarbonitrile precursors to direct the precursors tetramerization to be opposing subunits as described in Inorg. Chem. (1994),33, 1735-1740, Chemistry Letters (1992), 2031-2034 and Chemistry Letters (1992),1567-1570. These references, however, do not describe the synthesis of the novel phthalocyanine derivatives described herein using diiminoisoindoline precursors with or without axial ligands.
Preferred hybrid phthalocyanine derivatives have similar opposing subunits so that two different subunits comprise the structure. Particularly preferred hybrid phthalocyanine derivatives have similar opposing subunits on one axis and different opposing subunits on the other axis. The nature of the particularly preferred molecules is that red or blue shifted excitation or emission wavelengths and a longer Stokes shift can result because of the selection of the precursor molecules for the tetramerization reaction. For particularly preferred hybrid phthalocyanine derivatives, for example, the "donor" diphenyldiiminoisoindoline or the diiminoisoindoline precursors would contribute to 650 nm absorbance of the hybrid molecule, and thereby to the excitation of the hybrid molecule. The diphenyl phenyldiiminoisoindoline or the phenyldiiminoisoindoline precursors would act as an "electron transfer subunit" to the "acceptor subunit", which would be a dialkoxy or aryloxy phenyldiiminoisoindoline precursors, so that emission is dictated at the lowest energy by the acceptor subunit at about 850 nm. The nature of the "electron transfer subunit" is important because it is not desirable for this subunit to emit because then the desired emission of the acceptor subunit will not take place. Thus, the HOMO and LUMO character of the electron transfer subunit should be designed with reference to the donor and acceptor subunit molecules. The relationship of the energies of the HOMO and LUMO as they relate to excitation and emission are taught by Pariser et al, J. Chem. Phys. (1953), 21, 767-776, by Pople, Trans. Faraday Soc. (1953), 49, 1375-1385, by McHugh et al, Theoret. Chim. Acta (Berlin) (1972), 24, 346-370 and by Kobayashi et al, Inorg. Chem. (1994),33, 1735-1740, Chemistry Letters (1992), 2031-2041, Konami et al, Molecular Physics (1993), 80, 153-160.
Another application requires the hybrid molecule to have two excitation wavelengths, one at approximately 650 nm and another at about 680 nm with emission for both excitations at about 760 nm. Thus, the precursors responsible for the excitation could be a diiminoisoindoline for the 650 nm and a tetrafluorodiiminoisoindoline for the 680 nm excitations. The emitting subunit, which can also be used to direct the tetramerization reaction so that the emitting subunits are opposed in the molecule, can be a diphenyl phenyldiiminoisoindoline. The excitation and emission wavelengths of the resulting hybrid phthalocyanine derivative are thus generally representitive of the individual diiminoisoindoline precursors.
Yet another application requires excitation at about 650 nm and emission at about 750 nm. The precursors responsible for excitation and emission could be diiminoisoindoline and diphenyl phenyldiiminoisoindoline, respectively. The latter precursor also acts to direct the emitting subunits to be opposed.
In another application, a large extinction coefficient at the excitation wavelength is desired for excitation at about 650 nm. The emission wavelength should be at about 850 nm. The precursors responsible for excitation could be a diphenyldiiminoisoindoline, which would direct these subunits to be opposed and thereby two subunits would contribute to provide the desired extinction coefficient. A phenyldiiminoisoindoline derivative precursor could act as an electron transfer subunit and an alkoxy phenyldiiminoisoindoline precursor could be the acceptor with a characteristic emission at about 850 nm.
In another application, two emission wavelengths are desired from a compound which is excited at a single wavelength. The desired excitation is around 650 nm and the emission should be around 760 nm and 810 nm. The precursor responsible for excitation could be a tetrafluorodiiminoisoindoline or a tetrafluorobenzene-1,2-dicarbonitrile. The precursor responsible for emission could be a dibutoxyphenyldiiminoisoindoline or a 3,4-dibutoxy naphthalene-1,2-dicarbonitrile, respectively.
The resulting compounds are then incorporated into particles to yield particles which exhibit excitation wavelengths above about 600 nm and emission wavelengths above about 650 nm. One skilled in the art will also appreciate that water soluble hybrid phthalocyanine derivatives are valuable for coupling to proteins, polypeptides, nucleosides, nucleic acids and the like, for detecting their presence in biological fluids or for performing DNA probe or immunoassays.
Preferred particle sizes range from about 0.1 nm to 5000 nm and preferably from about 1 nm to 1000 nm. The choice of particle size should be related to the specific function for the label. The particle size may vary for a particular application. For example, in an immunoassay, if the label requires a more intense fluorescence for measuring very low concentrations of analytes, then one would employ larger particles because larger particles can incorporate more dye molecules. The small particle sizes (0.1-1 nm) may be employed in fluorescence polarization assays, as described for example, in U.S. Pat. Nos. 4,420,568, 4,476229 and 4,510,251, in in vitro visualization of cellular components or in in vivo imaging techniques.
The resulting fluorescent dye particles which exhibit the appropriate excitation and emission characteristics are further adsorbed or chemically reacted with various nucleic acids, nucleotides, proteins or peptides and the like which are required for a specific purpose. The adsorption of macromolecules to particles, particularly latex particles is well known to those skilled in the art and generally involves adsorption of the macromolecule at a temperature between 5.degree. C. and 50.degree. C. and at a pH which is below the pI of the molecule. For example, fluorescent particles exhibiting fluorescence energy transfer can be adsorbed with either antibodies for use in non-competitive immunoassays or ligand analogues for use in competitive immunoassays in reaction mixtures of the assays. In the case of non-competitive assays, the reaction mixture would include at least one target ligand and at least one class of fluorescent particles having bound thereto at least one receptor specific for target ligand, forming an antibody (fluorescent) conjugate. In the case of competitive assays, the reaction mixture will include at least one target ligand, at least one receptor specific to the target ligand, and at least one class of fluorescent particles, having bound thereto at least one ligand analogue, forming a ligand analogue (fluorescent) conjugate. The antibody conjugates bound to target ligands in the non-competitive reaction mixture and the ligand analogue conjugates not bound by receptors specific to the target ligands in the competitive reaction mixture can be bound to a solid phase consisting of receptors specific to another epitope of the target ligand of the target ligand-antibody conjugate complexes and of receptors specific to ligand analogues of the ligand analogue conjugates, respectively. The fluorescent conjugates unbound by the solid phase are removed and the fluorescence of the bound conjugates is measured. The measured fluorescence is related to the target ligand concentration. The various reagents described above can also be attached covalently to the latex particles. For example, antibodies or ligand analogues can be attached through amine or carboxylic acids to carboxylic acids or amines on the surface of the particles, respectively, to form stable amide linkages.
In the case of quantifying nucleic acids in samples, the novel compounds described in the instant invention are useful because of their brightness and because of the near infrared emission characteristics. In general, in designing an assay for a nucleic acid, one selects a probe molecule which is complementary to the nucleic acid to be quantified. The probe molecule is then labeled, generally covalently, with a signal generator. The signal generator can be a water soluble phthalocyanine derivative or hybrid phthalocyanine derivative or a particle with the appropriate dye system, which may exhibit fluorescence energy transfer or hybrid phthalocyanine derivatives or combinations of these compounds. The labelled probe molecule is then introduced into a biological sample suspected of containing the target nucleic acid, and the labelled probe sequence assembles with the target nucleic acid. The labelled probe/target nucleic acid can then be immobilized onto a surface which has immobilized another nucleic acid which is also complementary to the target nucleic acid. Conversely, the biological sample can be introduced to a surface which has immobilized a complementary nucleic acid for immobilization of the target nucleic acid. The labelled probe can then be introduced to the system for binding to the immobilized target molecule. The excess labelled probe is then washed away and the resultant fluorescent intensity is correlated with fluorescence intensity from a standard curve to arrive at a concentration of the nucleic acid in the sample.
Those skilled in the art will recognize that many variations of immunoassays and nucleic acid assays can be performed and the inventive teachings in the instant invention for the use of novel dye systems can be used to develop novel adaptations to existing technologies.
Those skilled in the art will appreciate that the novel fluorescent particles described herein have many uses in immunoassays, fluorescence microscopy, in vivo imaging, in vitro cancer therapy, nucleic acid assays, cell sorters and the like.
Experimental Section
Fluorescence measurements were performed on a Perkin-Elmer model LS 50B Luminescence Spectrometer for dyes emitting up to around 780 nm. In some instances, dyes emitting above 800 nm were measured according to Example 18. The fluorescence intensities are not corrected. Absorbance measurements were performed on a Hewlett Packard 8452A Diode Array Spectrophotometer.
EXAMPLE 1
Synthesis of Silicon Phthalocyanine Dihydroxide SiPc(OH).sub.2
A suspension of silicon phthalocyanine dichloride (1.83 g, 3.0 mmol) in pyridine (50 mL) and water (50 mL) was refluxed with stirring on an oil bath at 120.degree. C. for 18 hours. After cooling the dark blue solid product was filtered and the residue was washed with water (10 mL), acetone (5 mL) and then dried under vacuum to afford 1.71 g of the title compound.
EXAMPLE 2
Synthesis of Silicon Phthalocyanine bis(trihexylsilyloxide) (hereinafter sometimes referred to as PcSi trihexyl)
A suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol) in anhydrous pyridine (11 mL) containing chlorotrihexylsilane (733 .mu.L, 2.0 mmol) was refluxed on an oil bath at 130.degree. C. for 5 hours. The resulting purple solution was allowed to cool and was evaporated. The resulting slurry was treated with ice-cold hexane (2 mL) and the dark blue solid product was filtered, washed with ice-cold hexane (2 mL) and was dried under vacuum to yield 249 mg of crude product. The crude product in chloroform was purified on an Alumina column (Activity 1) equilibrated in hexane and the product was eluted with hexane/toluene (2/1, v/v) as a bright blue band. The solvent containing the product was evaporated to yield 69 mg of the title compound with a mp 171.degree. C. (lit mp 175.degree. C.).
EXAMPLE 3
Synthesis of Silicon Phthalocyanine bis�(10-carbomethoxydecyl) dimethylsilyloxide! (Hereinafter sometimes referred to as PcSi methyl ester)
To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol) in anhydrous pyridine (11 mL) was added (10-carbomethoxydecyl)dimethylchlorosilane (586 mg, 2 mmol) and the mixture was refluxed with stirring on an oil bath at 130.degree. C. for 5 hours. The dark blue solution was allowed to cool and the solvent was evaporated. The residue was purified on a Silica gel 60 .ANG. column equilibrated in hexane and the product eluted slowly as a blue band with toluene. The toluene fraction containing product was evaporated, hexane (10 mL) was added to the residue and the blue product was filtered, washed with hexane and dried to afford 105 mg of the title compound.
EXAMPLE 4
Synthesis of Silicon Phthalocyanine bis(dimethylvinylsilyloxide) (Hereinafter sometimes referred to as PcSi vinyl)
To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol) in anhydrous pyridine (11 mL) was added chlorodimethylvinylsilane (276 .mu.L, 2.0 mmol) and the mixture was refluxed with stirring on an oil bath at 130.degree. C. for 5 hours. The dark solution was allowed to cool and was evaporated. The residue was purified on a Silica gel 60 .ANG. column equilibrated in hexane and the product was eluted with toluene as a blue band. The eluate containing product was evaporated, the residue treated with hexane and the dark blue solid product was filtered, washed with hexane and was dried under vacuum to afford 7.5 mg of the title compound.
EXAMPLE 5
Synthesis of Silicon Phthalocyanine bis�(3-cyanopropyl) dimethylsilyloxide! (Hereinafter sometimes referred to as PcSi cyano)
To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol) in anhydrous pyridine (11 mL) was added chloro(3-cyanopropyl)-dimethylsilane (328 .mu.L, 2.0 mmol) and the mixture was refluxed with stirring on an oil bath at 130.degree. C. for 5 hours. The purple solution was allowed to cool and was evaporated. The residue was purified on a Silica gel 60 .ANG. column equilibrated in hexane. The column was washed with toluene and the product was eluted with toluene/isopropyl alcohol (90/10, v/v) as a bright blue band. The eluate containing product was evaporated under vacuum to afford 101 mg of the title compound with a mp>260.degree. C.
EXAMPLE 6
Synthesis of Silicon Phthalocyanine bis(dimethylpentafluoro-phenylsilyloxide) (Hereinafter sometimes referred to as PcSi pentafluoro)
To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol) in anhydrous pyridine (11 mL) was added chlorodimethylpentafluorophenylsilane (376 .mu.L, 2.0 mmol) and the mixture was refluxed with stirring on an oil bath at 130.degree. C. for 5 hours. The dark green solution was allowed to cool and was evaporated. The residue was purified on a Silica gel 60 .ANG. column equilibrated in hexane. The product was eluted with toluene as a dark blue band. The eluate containing the product was evaporated, the residue was treated with hexane (10 mL) and the dark blue solid product was filtered, washed with hexane and was dried under vacuum to afford 73 mg of the title compound.
EXAMPLE 7
Synthesis of Silicon 2,3-Naphthalocyanine Dihydroxide (Hereinafter sometimes referred to as NaPcSi hydroxide)
A suspension of silicon 2,3-naphthalocyanine dichloride (280 mg, 0.34 mmol) in pyridine (10 mL) and water (10 mL) was refluxed with stirring on an oil bath at 130.degree. C. for 24 hours. After cooling to room temperature, the dark green solid product was filtered and, the residue was washed, successively, with water (5 mL) and acetone (2 mL). The product was dried under vacuum to afford 217 mg of the title compound.
EXAMPLE 8
Synthesis of Silicon 2,3-Naphthalocyanine bis(dimethylvinylsilyloxide) (Hereinafter sometimes referred to as NaPcSi vinyl)
To a suspension of silicon 2,3-naphthalocyanine dihydroxide (87 mg, 0.11 mmol) in anhydrous dimethylformamide (1 mL) was added chlorodimethylvinylsilane (0.042 mL, 0.3 mmol), followed by imidazole (14 mg, 0.2 mmol). The mixture was stirred under argon at room temperature for 24 hours. The solvent was evaporated and the residue was purified on a Silica gel 60 .ANG. column which was equilibrated in hexane. The product was eluted with toluene as a green band. The toluene fraction containing the product was evaporated and the residue was treated with hexane. The dark green solid was filtered, washed with hexane and was dried under vacuum to afford 26 mg of the title compound.
EXAMPLE 9
Synthesis of Silicon 2,3-Naphthalocyanine bis(dimethylpentafluorophenylsilyloxide (Hereinafter sometimes referred to as NaPcSi pentafluoro)
To a suspension of silicon 2,3-naphthalocyanine dihydroxide (87 mg, 0.11 mmol) in anhydrous pyridine (5 ml) was added chlorodimethylpentafluorophenylsilane (0.188 ml, 1 mmol). The mixture was refluxed with stirring on an oil bath at 130.degree. C. for 5 hours. After cooling, the solvent was evaporated and the residue was purified on a Silica gel 60 .ANG. column which was equilibrated in hexane. The product was eluted with toluene as a green band. The toluene fraction containing the product was evaporated and the residue was treated with hexane. The dark green solid was filtered, washed with hexane and was dried under vacuum to afford 23 mg of the title compound.
EXAMPLE 10
General Procedures for the Preparation of Dye-loaded Latex Particles of Varying Sizes
The various dyes were loaded into latex particles of varying sizes according to the general procedures outlined below. Two procedures are described and involve swelling latex particles with aqueous solutions of either tetrahydrofuran or dimethylformamide prior to addition of the dye solutions. Latex particle sizes used range from 67 nm to 783 nm and one skilled in the art recognizes that smaller and larger particles can be used. The choice of the organic solvent used to swell the particles depends solely on the solubility of the various dyes in either solvent. Tables 1 and 2 of Example 15 below show the aqueous organic solvent system and the optimum dye concentration which were used for the loading into particles for each dye pair or for hybrid phthalocyanine derivatives, respectively, of a selected number of dyes. One skilled in the art recognizes that many changes can be made to these procedures to prepare particles with different degrees of fluorescence intensities and quenching by loading higher or lower amounts of dye in the particles and also by changing the ratios of each dye pair to the other. One skilled in the art also recognizes that similar techniques are useful for incorporation of dyes into latex particles, for example, as described in U.S. Pat. Nos. 4,199,363 and 4,368,258.
Surfactant-free polystyrene sulfate latex particles in sizes ranging from 67 nm to 783 nm and carboxyl-modified latex ("CML") particles ranging from 200 nm to 400 nm particles were obtained through Interfacial Dynamics Corp. Inc., Portland Oreg.
Method 1 Utilizing Tetrahydrofuran
Tetrahydrofuran (0.36 mL) was added, dropwise over a 5 minutes period, to a stirring solution of 1.6 mL of 2.5% solids of latex particles at room temperature. The latex suspension was stirred at room temperature for an additional 30 minutes to swell the latex. The dye solution (0.04 mL), which consists of one or more dyes at an appropriate concentration in tetrahydrofuran, was added dropwise over 5 minutes to the stirred latex solution, to give the loading dye concentration (in 2 mL volume) as indicated in Table 1. The latex-dye solution was stirred at room temperature for 30 minutes in the dark. The latex solution was then transferred to dialysis tubing (Spectra-por, 12-14,000 molecular weight cutoff, Spectrum, Houston, Tex.) and the dye-latex solutions were dialyzed against water for 12 to 15 hours at 4.degree. C. The dye-latex solutions were removed from dialysis and the % solids of the solutions was calculated from the final volume after dialysis and the starting solids concentration.
Method 2 Utilizing Dimethylformamide
Dimethylformamide (1.33 mL) was added, dropwise over a 5 minute period, to a stirring solution of 0.6 mL of 6.7% solids of latex particles at room temperature. The latex suspension was stirred at room temperature for an additional 30 minutes to swell the latex. The dye solution (0.07 mL), which consists of one or more dyes at an appropriate concentration in dimethylformamide, was added dropwise over 5 minutes to the stirred latex solution, to give the loading dye concentration (in 2 mL volume) as indicated in Table 1. The latex-dye solution was stirred at room temperature for 30 minutes in the dark. The latex solution was then transferred to dialysis tubing (Spectra-por, 12-14,000 molecular weight cutoff, Spectrum, Houston Tex.) and the dye-latex solutions were dialyzed against water for 12 to 15 hours at 4.degree. C. The dye-latex solutions were removed from dialysis and the % solids of the solutions was calculated from the final volume after dialysis and the starting solids concentration.
EXAMPLE 11
Effect of Varying Dye Loading Concentration on Fluorescence Intensity and Optimization of Fluorescence Intensity Latex Particles
The incorporation of dye into latex particles must be optimized in order to achieve the maximum fluorescence intensity and to minimize the degree of fluorescence quenching of the dye molecules. Fluorescence quenching can be significant because of the close proximity of the dye molecules in the particles. The PcSi vinyl was incorporated into 67 nm latex particles (polystyrene sulfate from Interfacial Dynamics Corp. (IDC), Inc., Portland, Oreg.) using method 1 (example 10) at various concentrations as indicated in the table below. The dye latex particles were diluted to 0.0019% solids in either water or tetrahydrofuran for each dye concentration. The solutions were excited at 350 nm and the emission at 680 nm was measured. The percent quenching in the particles is: (1-�fluorescence intensity in water divided by the intensity in the organic solvent!).times.100. The table below shows the fluorescence intensities as a function of dye loading concentrations and quenching for each condition.
______________________________________Loading Dye Concentration(mg/mL) Intensity (680 nm) Quenching (%)______________________________________0.01 420 410.025 489 650.05 492 730.075 401 760.1 338 830.15 197 870.3 91 900.9 34 96______________________________________
These results show that an optimum loading dye concentration gives the highest fluorescence intensities and the lowest quenching. In this case, a dye concentration of between 0.025 and 0.05 mg/mL in the loading solution gives the best intensity and the least quenching. Less dye than 0.025 mg/mL gives less intensity and less quenching because the spacing of the dyes begins to significantly increase and more dye than 0.05 mg/mL gives less intensity and more quenching because of the increased closeness of the dyes in the particles. This type of experiment illustrates the procedure for optimization of fluorescence intensity and for minimizing quenching.
EXAMPLE 12
Verification of Fluorescence Energy Transfer in Latex Particles
The latex particles which were incorporated with various dyes for energy transfer were diluted to 0.06% to 0.001% solids in water and either tetrahydrofuran or dimethylformamide and the solutions of equal solids concentrations were excited at wavelengths which corresponded to the approximate excitation maximum of the donor dye. The particles were diluted into organic solvents in order to liberate the dyes from the latex, and therefore, disrupt any energy transfer process between the dyes in the particles. The fluorescence of the solutions in water and organic solvent at the emission maximum of the acceptor dye or dyes were recorded and compared. Fluorescence energy transfer was defined as significant when the emission intensity of the acceptor was at least 5-fold higher in water than in the organic solvent.
EXAMPLE 13
Effect of Varying Donor Dye Concentration With Respect to Acceptor Dye Concentration in Latex Particles on the Fluorescence Intensity of the Particles
Meso-tetra-2-dimethylaminophenyl porphyrin was made as follows. To a stirring solution of meso-tetra-2-aminophenyl porphyrin (100 mg, 0.15 mmol) and 37% aqueous formaldehyde (500 .mu.L, 6.0 mmol) in tetrahydrofuran (2.5 mL was added sodium cyanoborohydride (114 mg, 1.8 mmol). The mixture was then treated with a glacial acetic acid (60 .mu.L) over 10 minutes and stirred at room temperature for 3 hours. More glacial acetic acid (60 .mu.L) was added and the mixture stirred a further 1 hour at room temperature. The mixture was evaporated and the residue was purified on a Silica gel 60 .ANG. column which was equilibrated in toluene. The product was eluted with toluene/1% isopropanol as a dark brown band. The fraction containing the product was evaporated and the ink-blue solid residue dried under vacuum to afford 85 mg of the title compound.
Meso-tetra-2-dimethylaminophenyl porphyrin (Tdap synthesized from the meso-tetra-2-aminophenyl porphyrin which was obtained through Porphyrin Products, Inc. Logan, Utah) and PcSi vinyl (example 4) were incorporated into 67 nm latex particles (polystyrene sulfate latex from Interfacial Dynamics Inc., Portland, Oreg.) using the tetrahydrofuran method 1 of example 10. The molar ratio of the Tdap to the PcSi vinyl varied from 1/1 to 2/1 to 6/1 in the latex loading solutions while maintaining a constant mass (0.1 mg/mL) of PcSi vinyl in each solution. The dialyzed particles were diluted to 0.0019% solids in water and the fluorescence intensity at 680 nm of the PcSi vinyl was measured as a function of excitation wavelength between 350 nm and 470 nm. The excitation maximum of the Tdap is 430 nm and of the PcSi vinyl is 350 nm. The emission maximum of the Tdap is 650 nm. The table below shows the results.
______________________________________ Fluorescence Intensity atTdap/PcSi vinyl Excitation .lambda. (nm) 680 nm______________________________________1/1 350 4901/1 430 831/1 450 381/1 470 112/1 350 5802/1 430 8302/1 450 4602/1 470 2206/1 350 6006/1 430 18006/1 450 8006/1 470 200______________________________________
These results show that as the molar ratio of donor to acceptor in the latex particles increases from 1/1 to 6/1, the energy transfer, as measured by the fluorescence intensity of the acceptor dye, becomes significantly more efficient. There was no observable emission of the Tdap dye in the particles at the emission maximum of 650 nm suggesting that the energy transfer is very efficient. The data indicates that the fluorescence intensity of the latex particles, generated through an energy transfer pathway, is affected by the "light gathering" capability of the donor dye. Thus, optimization of the fluorescence intensity of the latex particles should involve changing the molar ratio of donor to acceptor.
EXAMPLE 14
Effect of Incorporation of Different Dyes on Ouenching and Fluorescence Intensity of Latex Particles
Five different silicon phthalocyanines, synthesized as described in examples 2-6, were incorporated into 67 nm surfactant-free, polystyrene latex particles (Interfacial Dynamics Corp. Inc. Portland, Oreg.) in sets of 1, 3 or 5 dyes according to the following methods. Each silicon phthalocyanine derivative had maximum excitation and emission wavelengths at 350 nm and 680 nm, respectively. After preparation of each dye-latex, each suspension was diluted to 0.057% solids in either water or tetrahydrofuran. The dye-latex solutions were excited at 350 nm and the fluorescence intensity at 680 nm was measured. The intensity of fluorescence in water divided by the intensity of fluorescence in tetrahydrofuran minus 1 is the degree of quenching of the dyes in the latex particles.
Preparation of One Phthalocyanine Dye in Latex
A solution of PcSi pentafluoro dye (0.02 mg) in tetrahydrofuran (0.1 mL) was added dropwise over 5 minutes to a stirred 2% solids solution of latex particles (1.0 mL). The latex suspension was stirred at room temperature for 6 hours, then transferred to dialysis tubing (Spectra-por, 12-14,000 molecular weight cutoff, Spectrum, Houston, Tex.) and the dye-latex solution was dialyzed against water for 12-15 hours at 4.degree. C. The dye-latex solution was removed from dialysis and the solids concentration was adjusted to 1.6%.
Preparation of Three Phthalocyanine Dyes in Latex
A solution which consists of PcSi pentafluoro, PcSi trihexyl and PcSi cyano dyes in equimolar amounts to total 0.02 mg dye in tetrahydrofuran (0.1 mL), was added drop-wise over 5 minutes to a stirred 2% solids solution of latex particles (1.0 mL). The latex suspension was stirred at room temperature for 6 hours, then transferred to dialysis tubing (Spectra-por, 12-14,000 molecular weight cutoff, Spectrum, Houston, Tex.) and the dye-latex solution was dialyzed against water for 12-15 hours at 4.degree. C. The dye-latex solution was removed from dialysis and the solids concentration was adjusted to 1.6%.
Preparation of Five Phthalocyanine Dyes in Latex
A solution which consists of PcSi pentafluoro, PcSi trihexyl, PcSi cyano, PcSi vinyl and PcSi methyl ester dyes in equimolar amounts to total 0.02 mg dye in tetrahydrofuran (0.1 mL), was added dropwise over 5 minutes to a stirred 2% solids solution of latex particles solution (1.0 mL). The latex suspension was stirred at room temperature for 6 hours, then transferred to dialysis tubing (Spectra-por, 12-14,000 molecular weight cutoff, Spectrum, Houston, Tex.) and the dye-latex solution was dialyzed against water for 12-15 hours at 4.degree. C. The dye-latex solutions were removed from dialysis and the % solids concentration was adjusted to 1.6%.
The table that follows illustrates the results of the fluorescence experiments.
______________________________________Dyes Entrapped Intensity % Quenching______________________________________1 413 723 561 565 747 49______________________________________
The data show that as the number of different dyes entrapped into the latex goes from 1 to 3 to 5, the fluorescence intensity increases because the quenching in the particles decreases.
EXAMPLE 15
Preparation and Characterization of Fluorescence Energy Transfer Dye Latex (Table 1) and Fluorescent Latex Incorporating Hybrid Phthalocyanine Derivatives (Table 2)
A variety of fluorescent energy transfer latexes were prepared with various donor and acceptor dye molecules. Table 1 shows the loading concentrations of the respective donor and acceptor dyes, the mole ratio of the donor and acceptor dyes, the dye loading solvent system as described in Example 10 and the excitation and emission wavelengths and the fluorescence intensity for each particle size at the specified solids concentration. For some of the energy transfer latexes, the same dye pair was incorporated into different diameter latexes. The fluorescence energy transfer efficiency of the entries is greater than 80%. The dye system represented in line 56 is a fluorescence energy transfer compound (FET compound) so that the donor and acceptor pair reside in the molecule before incorporation into latex.
Table 2 shows the characteristics of latex particles incorporated with hybrid phthalocyanine derivatives as described in Example 10 and the fluorescence intensity at the specified solids concentration.
TABLE 1__________________________________________________________________________ MOLE SOLVENT LOADING LOADING DONOR: SYSTEM EMISSION CONC. CONC. MOLE (LATEX INTENSITY MAXIMUMDONOR DYE (mg/mL) ACCEPTOR DYE (mg/mL) ACCEPTOR SIZE) (% SOLID) (EXCIT.)__________________________________________________________________________1. trans-4-�4-(Dibutyl amino) 0.12 Silicon phthalocyanine 0.1 2:1 THF 340 679 nmstyryl!-1-methyl pyridinium iodide mg/mL bis(dimethylvinylsilyl- mg/mL (0.067 (0.0019%) (475 nm) oxide) .mu.m)2. trans-4-�4-(Dibutyl amino) 0.1 Silicon 2,3-naphthalo- 0.23 1:1 DMF 347 789 nmstyryl!-1-methyl pyridinium iodide mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (475 nm) vinylsilyloxide) .mu.m)3. trans-4-�4-(Dibutyl amino) 0.1 1,1'-Dihexyl-3,3,3',3'- 0.144 1:1 DMF 688 688 nmstyryl!-1-methyl pyridinium iodide mg/mL tetramethylindodicarbo- mg/mL (0.067 (0.057%) (645 nm) cyanine iodide .mu.m)4. Meso-tetra-2-aminophenyl 0.18 Silicon phthalocyanine 0.1 2:1 THF 1000 679 nmporphine mg/mL bis(dimethylvinylsilyl- mg/mL (0.202 (0.00095%) (420 nm) oxide) .mu.m)5. Meso-tetra-2-aminophenyl 0.1 1,1'-Dihexyl-3,3,3',3'- 0.098 1:1 DMF 157 676 nmporphine mg/mL tetramethylindodicarbo- mg/mL (0.067 (0.0019%) (645 nm) cyanine iodide .mu.m)6. Meso-tetra-2- 0.21 Silicon phthalocyanine 0.1 2:1 THF 209 679 nmdimethylaminophenyl porphine mg/mL bis(dimethylvinyl- mg/mL (0.412 (0.00095%) (430 nm) silyloxide) .mu.m)7. 3-Ethyl-3'-ethyl carboxyethyl- 0.056 Silicon 2,3-naphthalo- 0.25 4:1 DMF 289 785 nmthiadicarbocyanine iodide mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (650 nm) vinylsilyloxide) .mu.m)8. 1,1'-Dioctadecyl-3,3,3,3',3'- 0.036 Silicon 2,3-naphthalo- 0.013 4:1 DMF 324 787 nmtetramethyl-indodicarbocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (650 nm)perchlorate vinylsilyloxide) .mu.m)9. 1,1'-Diethyl-3,3,3',3'- 0.078 Silicon 2,3-naphthalo- 0.025 6:1 DMF 723 787 nmtetramethylindodi-carbocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (635 nm)iodide vinylsilyloxide) .mu.m)10. 1,1'-Dihexyl-3,3,3',3'- 0.094 Silicon 2,3-naphthalo- 0.025 6:1 DMF 907 783 nmtetramethylindodicarbocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (635 nm)iodide vinylsilyloxide) .mu.m)11. 3,3'-Diethyl 0.013 Silicon 2,3-naphthalo- 0.025 1:1 DMF 12 788 nmthiadicarbocyanine iodide mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (650 nm) vinylsilyloxide) .mu.m)12. 3,3'-Dipropyl 0.013 Silicon 2,3-naphthalo- 0.025 1:1 DMF 65 788 nmthiadicarbocyanine iodide mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (660 nm) vinylsilyloxide) .mu.m)13. 1,9-Dimethyl-methylene 0.008 Silicon 2,3-naphthalo- 0.025 1:1 DMF 57 788 nmblue, chloride mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (650 nm) vinylsilyloxide) .mu.m)14. N,N'-Di(3-trimethyl- 0.013 Silicon 2,3- 0.025 1:1 DMF 63 788 nmammoniumpropyl) thia- mg/mL naphthalocyanine mg/mL (0.067 (0.057%) (650 nm)dicarbocyanine tribromide bis(dimethylvinyl- .mu.m) silyloxide)15. 1,1',3,3,3',3'-Hexamethyl- 0.012 Silicon 2,3-naphthalo- 0.025 1:1 DMF 33 788 nmindo-tricarbocyanine perchlorate mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (650 nm) vinylsilyloxide) .mu.m)16. N-(3-Triethyl- 0.014 Silicon 2,3-naphthalo- 0.025 1:1 DMF 55 788 nmammoniumpropyl)-4-(4-(p- mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (500 nm)dibutylaminophenyl) butadienyl) vinylsilyloxide) .mu.m)pyridium, dibromide17. 1,1',3,3,3',3'-Hexamethyl- 0.015 Silicon 2,3-naphthalo- 0.025 1:1 DMF 8 788 nm4,4'-5,5'-dibenzo-2,2'-indo- mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (650 nm)tricarbocyanine perchlorate vinylsilyloxide) .mu.m)18. Fluoroscein 0.264 Silicon phthalocyanine 0.1 6:1 THF 517 683 nm mg/mL bis(dimethylvinyl- mg/mL (0.067 (0.057%) (485 nm) silyloxide) .mu.m)19. Chlorophyll B 0.087 Silicon 2,3-naphthalo- 0.025 4:1 THF 72 783 nm mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (440 nm) vinylsilyloxide) .mu.m)20. Chlorophyll B 0.244 Silicon phthalocyanine 0.1 2:1 THF 140 679 nm mg/mL bis(dimethylvinyl- mg/mL (0.067 (0.0019%) (440 nm) silyloxide) .mu.m)21. trans-4-�4-(Dibutyl 0.181 Silicon phthalocyanine 0.07 4:1:1 THF 300 681 nmamino)styryl!-1-methyl pyridinium mg/mL bis(dimethylpenta- mg/mL (0.067 (0.0019%) (475 nm)iodide fluorophenylsilyloxide) .mu.m) + Silicon phthalocyanine 0.05 bis(dimethylvinylsilyl- mg/mL oxide)22. trans-4-�4-(Dibutyl amino) 0.072 Silicon phthalocyanine 0.04 4:1:1:1 THF 206 681 nmstyryl!-1 -methyl pyridinium iodide mg/mL bis(trihexylsilyloxide) mg/mL (0.067 (0.0019%) (475 nm) + .mu.m) Silicon phthalocyanine 0.04 bis(dimethylpentafluoro- mg/mL phenylsilyloxide) + Silicon phthalocyanine 0.03 bis(dimethylvinylsilyl- mg/mL oxide)23. 3-Ethyl-3'-carboxyethylthia- 0.013 Silicon 2,3-naphthalo- 0.025 1:1 DMF 76 788 nmdicarbocyanine iodide mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (625 nm) vinylsilyloxide) .mu.m)24. 3-Ethyl-3'-ethyl-carboxy- 0.013 Silicon 2,3-naphthalo- 0.025 1:1 DMF 135 788 nmethyloxathiadicarbocyanine iodide mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (630 nm) vinylsilyloxide) .mu.m)25. 3,3'-Diethylthia- 0.013 Silicon 2,3-naphthalo- 0.025 1:1 DMF 59 787 nmdicarbocyanine iodide mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (660 nm) vinylsilyloxide) .mu.m)26. 3,3'-Diethyloxa- 0.012 Silicon 2,3-naphthalo- 0.025 1:1 DMF 57 787 nmdicarbocyanine iodide mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (590 nm) vinylsilyloxide) .mu.m)27. 1,1'-Dihexyl-3,3,3',3'- 0.094 Silicon 2,3-naphthalo- 0.025 6:1:2 DMF 127 788 nmtetramethyl-indodicarbocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.431 (0.057%) (650 nm)iodide vinylsilyloxide) .mu.m CML) + Silicon naphthalocyanine 0.05 bis(dimethylethyl- mg/mL maleimidosilyloxide)28. 1,1'-Dihexyl-3,3,3',3'- 0.094 Silicon 2,3-naphthalo- 0.025 6:1:2 DMF 193 788 nmtetramethyl-indodicarbocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.431 (0.057%) (635 nm)iodide vinylsilyloxide) .mu.m CML) + Silicon phthalocyanine 0.05 bis(dimethylethyl- mg/mL maleimidosilyloxide)29. 1,1'-Dihexyl-3,3,3',3'- 0.03 Silicon 2,3-naphthalo- 0.05 1:1 DMF 275 788 nmtetramethyl-indodicarbocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.431 (0.057%) (650 nm)iodide hexylvinylsilyloxide) .mu.m CML)30. 1,1'-Dihexyl-3,3,3',3'- 0.1 Silicon 2,3 naphthalo- 0.2 1:1 DMF 163 798 nmtetramethyl-indodicarbocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.431 (0.057%) (650 nm)iodide triphenylsilyloxide) .mu.m CML)31. 1,1'-Dihexyl-3,3,3',3'- 0.09 Silicon naphthalocyanine 0.05 4:1 DMF 153 790 nmtetramethyl-indodicarbocyanine mg/mL bis(dimethylretinol) mg/mL (0.431 (0.057%) (650 nm)iodide .mu.m CML)32. 1,1',3,3,3',3'-Hexamethyl- 0.216 Silicon 2,3-naphthalo- 0.1 4:1 DMF 0.4 788 nmindotricarbocyanine perchlorate mg/mL cyanine bis(dimethyl- mg/mL (0.431 (0.00057%) (635 nm) vinylsilyloxide) .mu.m CML)33. 1,1'-Dihexyl-3,3,3',3'- 0.512 1,1',3,3,3',3'- 0.1 4:1 DMF 0.9 776 nmtetramethylindo-dicarbocyanine mg/mL Hexamethylindotri- mg/mL (0.431 (0.00057%) (635 nm)iodide carbocyanine perchlorate .mu.m CML)34. Lithium tetraacetylide boron 0.16 Silicon 2,3-naphthalo- 0.1 4:1 DMF 22 788 nmcomplex of 1,1'-Dihexyl- mg/mL cyanine bis(di-methyl- mg/mL (0.216 (0.00057%) (635 nm)3,3,3',3'-tetramethylindo- hexylvinylsilyloxide) .mu.m CML)dicarbocyanine iodide35. Silicon phthalocyanine 0.334 Silicon 2,3-naphthalo- 0.1 10:1 DMF 1 800 nmbis(di-methylvinylsilyloxide) mg/mL cyanine bis(di-methyl- mg/mL (0.216 (0.00057%) (650 nm) hexylvinylsilyloxide) .mu.m CML)36. 1,1',3,3,3',3'- 0.23 Silicon 2,3-naphthalo- 0.1 10:1 DMF 0.4 780 nmHexamethylindotri-carbocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (635 nm)perchlorate hexylvinylsilyloxide) .mu.m CML)37. 1,1',3,3,3',3'- 0.19 Silicon octaethoxy 2,3- 0.1 10:1 DMF 0.7 780 nmHexamethylindotri-carbocyanine mg/mL naphthalocyanine bis(di- mg/mL (0.216 (0.00057%) (635 nm)perchlorate methylhexylvinyl- .mu.m CML) silyloxide)38. Oxazine 1 perchlorate 0.01 Silicon 2,3-naphthalo- 0.025 1:1 DMF 291 788 nm mg/mL cyanine bis(dimethyl- mg/mL (0.067 (0.057%) (650 nm) vinylsilyloxide) .mu.m)39. 3,3'-Dipropyl- 0.232 Silicon 2,3-naphthalo- 0.1 4:1 DMF 0.4 788 nmthiadicarbocyanine iodide mg/mL cyanine bis(dimethyl- mg/mL (0.431 (0.00057%) (635 nm vinylsilyloxide) .mu.m CML)40. Copper tetra-tert-butyl 0.72 Silicon 2,3-naphthalo- 0.1 1:1 DMF 0.2 788 nmphthalocyanine mg/mL cyanine bis(di- mg/mL (0.216 (0.00057%) (650 nm) methylhexylvinyl- .mu.m CML) silyloxide)41. (E,E)-3,5-bis-(4-phenyl-1,3- 0.16 Silicon 2,3-naphthalo- 0.1 4:1 DMF 42 785 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene hexylvinylsilyloxide) .mu.m CML)42. Aluminum tetra-tert-butyl 0.28 Silicon 2,3-naphthalo- 0.1 4:1 THF 0.5 788 nmphthalocyanine hydroxide mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (650 nm) hexylvinylsilyloxide) .mu.m CML)43. Aluminum 0.29 Silicon 2,3-naphthalo- 0.1 4:1 DMF 0.1 788 nmtetra-tert-butylphthalocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (650 nm)chloride hexylvinylsilyloxide) .mu.m CML)44. (E,E)-3,5-bis-(4-phenyl-1,3- 0.14 Aluminum octabutoxy- 0.1 4:1 THF 1.8 774 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL phthalocyanine mg/mL (0.216 (0.00057%) (650 nm)3a,4a-diazo-s-indacene triethylsilyloxide .mu.m CML)45. Iron phthalocyanine 0.26 Silicon 2,3-naphthalo- 0.1 4:1 THF 0.3 788 nmbis(tert-butyl isocyanide) mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm) hexylvinylsilyloxide) .mu.m CML)46. (E,E)-3,5-bis-(4-phenyl-1,3- 0.16 Octabutoxy- 0.1 4:1 THF 0.7 783 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL phthalocyanine mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene .mu.m CML)47. (E,E)-3,5-bis-(4-phenyl-1,3- 0.15 Silicon 2,3-naphthalo- 0.1 4:1 THF 16.9 783 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene phenylpentafluoro- .mu.m CML) silyloxide)48. (E,E)-3,5-bis-(4-phenyl-1,3- 0.19 Silicon 2,3-naphthalo- 0.1 4:1 THF 31.5 783 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene vinylsilyloxide) .mu.m CML)49. (E,E)-3,5-bis-(4-phenyl-1,3- 0.15 Silicon 2,3-naphthalo- 0.1 4:1 THF 13.1 783 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene bis(diphenylvinyl- .mu.m CML) silyloxide)50. (E,E)-3,5-bis-(4-phenyl-1,3- 0.15 Silicon 2,3-naphthalo- 0.1 4:1 THF 4.7 783butadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene maleimidoethoxysilyl- .mu.m CML) oxide)51. (E,E)-3,5-bis-(4-phenyl-1,3- 0.14 Silicon 2,3-naphthalo- 0.1 4:1 THF 11.7 783 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene silyl-oxide-trans-stilbene) .mu.m CML)52. (E,E)-3,5-bis-(4-phenyl-1,3- 0.12 Silicon 2,3-naphthalo- 0.1 4:1 THF 22.3 783 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(tri-deca- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene fluoro-1,1,2,2-tetra- .mu.m CML) hydrooctyl-1-dimethyl- silyloxide)53. (E,E)-3,5-bis-(4-phenyl-1,3- 0.12 Silicon 2,3-naphthalo- 0.1 4:1 THF 16.1 783 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene retinol) .mu.m CML)54. Germanium tetra-tert-butyl 0.3 Silicon 2,3-naphthalo- 0.1 4:1 THF 1.3 783 nmphthalocyanine dihydroxide mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm) hexylvinylsilyloxide) .mu.m CML)55. Germanium tetra-tert-butyl 0.3 Silicon 2,3-naphthalo- 0.1 4:1 THF 0.6 783 nmphthalocyanine dichloride mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)5 hexylvinylsilyloxide) .mu.m CML)56. Silicon phthalocyanine bis 0.15 Silicon phthalocyanine THF 209 681 nm(maleimide-fluoroscein) mg/mL bis (maleimide- (0.067 (0.0019%) (470 nm)FET COMPOUND fluoroscein) .mu.m) FET COMPOUND57. 3,3'-Diethylthia- 0.57 5,5'-Dichloro-1,1'- 0.1 4:1 DMF -0.048 832 nmtricarbocyanine iodide mg/mL diphenylamino-3,3'- mg/mL (0.216 (0.00057%) (670 nm) diethyl-10,12-ethylene- .mu.m CML) thiatricarbocyanine iodide58. 1,1',3,3,3',3'- 0.61 5,5'-Dichloro-1,1'- 0.1 4:1 DMF -0.149 832 nmHexamethylindotri-carbocyanine mg/mL diphenylamino-3,3'- mg/mL (0.216 (0.00057%) (670 nm)perchlorate diethyl-10,12-ethylene- .mu.m CML) thiatricarbocyanine iodide59. 1,1',3,3,3',3'-Hexamethyl- 0.51 5,5'-Dichloro-1,1'- 0.1 4:1 DMF -0.046 832 nm4,4',5,5'-dibenzo-2,2'-indo- mg/mL diphenylamino-3,3'- mg/mL (0.216 (0.00057%) (670 nm)tricarbocyanine perchlorate diethyl-10,12-ethylene- .mu.m CML) thiatricarbocyanine iodide60. 1,1'-Dihexyl-3,3,3',3'- 0.23 Silicon 2,3-naphthalo- 0.1 4:1 DMF -14.12 783 nmtetramethyl-indodicarbocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)iodide hexylvinylsilyloxide) .mu.m CML)61. (E,E)-3,5-bis-(4-phenyl-1,3- 0.16 Silicon 2,3-naphthalo- 0.1 4:1 DMF -5.00 783 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene hexylvinylsilyloxide) .mu.m CML)62. (E,E)-3,5-bis-(4-phenyl-1,3- 0.26 Silicon octaethoxy2,3- 0.1 4:1 DMF -2.74 858 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL naphthalocyanine bis(di- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene methylhexylvinylsilyl- .mu.m CML) oxide)63. (E,E)-3,5-bis-(4-phenyl-1,3- 0.32 Octabutoxy- 0.1 4:1 DMF -4.07 762 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL phthalocyanine mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene .mu.m CML)64. (E,E)-3,5-bis-(4-phenyl-1,3- 0.28 Octabutoxy- 0.1 4:1 DMF -1.76 772 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL naphthalocyanine mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene .mu.m CML)65. 1,1'-Dihexyl-3,3,3',3'- 0.19 Silicon octaethoxy2,3- 0.1 4:1 DMF -0.712 858 nmtetramethylindo-dicarbocyanine mg/mL naphthalocyanine bis(di- mg/mL (0.216 (0.00057%) (670 nm)iodide methylhexylvinyl- .mu.m CML) silyloxide)66. 3,3'-Diethylthia- 0.16 Silicon octaethoxy2,3- 0.1 4:1 DMF -0.058 858 nmtricarbocyanine iodide mg/mL naphthalocyanine bis(di- mg/mL (0.216 (0.00057%) (670 nm) methylhexylvinyl- .mu.m CML) silyloxide)67. 1,1',3,3,3',3'- 0.15 Silicon octaethoxy 2,3- 0.1 4:1 DMF -0.141 858 nmHexamethylindotri-carbocyanine mg/mL naphthalocyanine bis(di- mg/mL (0.216 (0.00057%) (670 nm)perchlorate methylhexylvinyl- .mu.m CML) silyloxide)68. 1,1',3,3,3',3'-Hexamethyl- 0.19 Silicon octaethoxy 2,3- 0.1 4:1 DMF -0.058 858 nm4,4',5,5'-dibenzo-2,2'-indo- mg/mL naphthalocyanine bis(di- mg/mL (0.216 (0.00057%) (670 nm)tricarbocyanine perchlorate methylhexylvinyl- .mu.m CML) silyloxide)69. (E,E)-3,5-bis-(4-phenyl-1,3- 0.2 Silicon octaethoxy 2,3- 0.15 4:1 THF -2.720 858 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL naphthalocyanine bis(di- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene methylhexylvinyl- .mu.m CML) silyloxide)70. (E,E)-3,5-bis-(4-phenyl-1,3- 0.16 Silicon 2,3-naphthalo- 0.1 4:1:1 THF -2.38 858 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene hexylvinylsilyloxide) + .mu.m CML) Silicon octaethoxy 2,3- naphthalocyanine bis(di- 0. 12 methylhexylvinylsilyl- mg/mL oxide)71. Silicon phthalocyanine 0.36 5,5'-Dichloro-1,1'- 0.1 4:1 THF -8.10 832 nmbis(di-methylvinylsilyloxide) mg/mL diphenylamino-3,3'- mg/mL (0.216 (0.00057%) (670 nm) diethyl-10,12-ethylene- .mu.m CML) thiatricarbocyanine perchlorate72. Tetrakis(4-cumyl-phenoxy) 0.48 Silicon 2,3-naphthalo- 0.1 4:1 THF -0.397 783 nmphthalocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm) hexylvinylsilyloxide) .mu.m CML)73. Tetrakis(4-cumyl-phenoxy) 0.68 5,5'-Dichloro-1,1'- 0.1 4:1 THF -0.128 832 nmphthalocyanine mg/mL diphenylamino-3,3'- mg/mL (0.216 (0.00057%) (670 nm) diethyl-10,12-ethylene- .mu.m CML) thiatricarbocyanine perchlorate74. Tetrakis(phenylthio) 0.34 Silicon 2,3-naphthalo- 0.1 4:1 THF -0.374 788 nmphthalocyanine mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm) hexylvinylsilyloxide) .mu.m CML)75. Tetrakis(phenylthio) 0.28 5,5'-Dichloro-1,1'- 0.1 4:1 THF -0.109 832 nmphthalocyanine mg/mL diphenylamino-3,3'- mg/mL (0.216 (0.00057%) (670 nm) diethyl-10,12-ethylene- .mu.m CML) thiatricarbocyanine perchlorate76. (E,E)-3,5-bis-(4-phenyl-1,3- 0.24 Tin octabutoxy 2,3- 0.1 4:1 THF -1.724 >900 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL naphthalocyanine mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene dichloride .mu.m CML)77. Tetrakis 0.36 Tin octabutoxy 2,3- 0.1 4:1 THF -0.162 >900 nm(4-cumylphenoxy) phthalocyanine mg/mL naphthalocyanine mg/mL (0.216 (0.00057%) (670 nm) dichloride .mu.m CML)78. Tetrakis(phenylthio) 0.26 Tin octabutoxy 2,3- 0.1 4:1 THF -0.061 >900 nmphthalocyanine mg/mL naphthalocyanine mg/mL (0.216 (0.00057%) (670 nm) dichloride .mu.m CML)79. Germanium tetra-tert-butyl 0.42 5,5'-Dichloro-1,1'- 0.1 4:1 THF -0.109 >900 nmphthalocyanine dihydroxide mg/mL diphenylamino-3,3'- mg/mL (0.216 (0.00057%) (670 nm) diethyl-10,12-ethylene- .mu.m CML) thiatricarbocyanine perchlorate80. Germanium tetra-tert-butyl 0.22 Tin octabutoxy 2,3- 0.1 4:1 THF -0.045 >900 nmphthalocyanine dihydroxide mg/mL naphthalocyanine mg/mL (0.216 (0.00057%) (670 nm) dichloride .mu.m CML)81. Germanium tetra-tert-butyl 0.2 Tin octabutoxy 2,3- 0.1 4:1 THF -0.042 >900 nmphthalocyanine dihydroxide mg/mL naphthalocyanine bis mg/mL (0.216 (0.00057%) (670 nm) (triethylsilyloxide) .mu.m CML)82. Germanium tetra-tert-butyl 0.42 5,5'-Dichloro-1,1'- 0.1 4:1 THF -0.081 832 nmphthalocyanine dichloride mg/mL diphenylamino-3,3'- mg/mL (0.216 (0.00057%) (670 nm) diethyl-10,12-ethylene- .mu.m CML) thiatricarbocyanine perchlorate83. Germanium tetra-tert-butyl 0.22 Tin octabutoxy 2,3- 0.1 4:1 THF -0.052 >900 nmphthalocyanine dichloride mg/mL naphthalocyanine mg/mL (0.216 (0.00057%) (670 nm) dichloride .mu.m CML)84. Germanium tetra-tert-butyl 0.2 Tin octabutoxy 2,3- 0.1 4:1 THF -0.050 >900 nmphthalocyanine dichloride mg/mL naphthalocyanine bis mg/mL (0.216 (0.00057%) (670 nm) (triethylsilyloxide) .mu.m CML)85. (E,E)-3,5-bis-(4-phenyl-1,3- 0.16 Silicon 2,3-naphthalo- 0.1 4:1:1 THF -0.315 858 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene hexylvinylsilyloxide) + .mu.m CML) 5,5'-Dichloro-1,1'- diphenylamino-3,3'- 0.072 diethyl-10,12-ethylene- mg/mL thiatricarbocyanine perchlorate86. (E,E)-3,5-bis-(4-phenyl-1,3- 0.24 5,5'-Dichloro-1,1'- 0.1 4:1 THF -2.230 832 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL diphenylamino-3,3'- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene diethyl-10,12-ethylene- .mu.m CML) thiatricarbocyanine perchlorate87. 1,1'-Dihexyl-3,3,3',3'- 0.34 5,5'-Dichloro-1,1'- 0.1 4:1 THF -0.545 823 nmtetramethyl-indodicarbocyanine mg/mL diphenylamino-3,3'- mg/mL (0.216 (0.00057%) (670 nm)iodide diethyl-10,12-ethylene- .mu.m CML) thiatricarbocyanine perchlorate88. (E,E)-3,5-bis-(4-phenyl-1,3- 0.16 Silicon 2,3-naphthalo- 0.07 4:1:1 THF 49 783 nmbutadienyl)-4,4-difluoro-4-bora- mg/mL cyanine bis(dimethyl- mg/mL (0.216 (0.00057%) (670 nm)3a,4a-diazo-s-indacene hexylvinylsilyloxide) + .mu.m CML) Silicon 2,3-naphthalo- cyanine bis(dimethyl- 0.07 pentafluorophenyl-silyl- mg/mL oxide)__________________________________________________________________________
TABLE 2__________________________________________________________________________ LOADING CONC. SOLVENT LATEX EMISSIONHYBRID COMPOUND (mg/mL) SYSTEM SIZE % SOLID INTENSITY MAXIMUM EXCITATION__________________________________________________________________________1. Silicon �di(1,6-diphenylnaphthalocyanine)! 2.0 THF 0.216 0.00057% 50 760 nm 650 nmdiphthalocyanine bis(dimethylhexyl- mg/mL .mu.m CMLvinylsilyloxide)2. Silicon �di(1,6-diphenylnaphthalocyanine)! 2.0 THF 0.216 0.00057% 0.7/0.5 765 nm/ 650 nmtetrafluorophthalocyanine mg/mL .mu.m CML 825 nmphthalocyaninebis(dimethylhexylvinylsilyloxide)3. Silicon �di(1,6-diphenylnaphthalocyanine)! 1.5 THF 0.216 0.00057% 0.5/0.3 770 nm/ 650 nmtetrafluorophthalocyanine mg/mL .mu.m CML 839 nmphthalocyaninebis(dimethylpentafluorophenylsilyloxide)4. Silicon �di(1,6-diphenylnaphthalocyanine)! 0.1 THF 0.216 0.00057% 0.2 775 nm 650 nmdiphthalocyanine bis(dimethylpentafluoro- mg/mL .mu.m CMLphenylsilyloxide)5. Silicon �di(1,6-diphenylnaphthalocyanine)! 1.5 THF 0.216 0.00057% 19 758 nm 650 nmdi(tert-butyl-phthalocyanine) bis(dimethyl- mg/mL .mu.m CMLhexylvinylsilyloxide)__________________________________________________________________________
EXAMPLE 16
Adsorption of Anti-Human Chorionic Gonadotropin (hCG) Antibody to Latex Particles
A typical example of the adsorptions of an antibody to dyed latex particles, prepared as described in Example 10, and of a complementary antibody to undyed latex particles, both of which can be used in a sandwich assay for hCG, is outlined below. Those skilled in the art will recognize that various techniques are available to adsorb or to covalently couple proteins, peptides, ligand analogues nucleotides and nucleic acids to latex particles. A solution of dye latex (0.1 mL, 2% solids, 412 nm; entry 10, Table 1) was added quickly while vortexing to a solution of anti-.beta. hCG monoclonal antibody (0.2 mL, 6.6 mg/mL; Applied Biotech Inc., San Diego, Calif.) in 20 mM sodium borate/150 mM sodium chloride, pH 8.2. A solution of 0.1M potassium citrate, pH 3, (0.04 mL) was added quickly while vortexing to the antibody latex solution at room temperature and the pH of the resulting solution was 3.5. The solution incubated at room temperature for 5 minutes, then a solution of 2M potassium borate, pH 9.7 (0.025 mL) was added quickly while vortexing to bring the pH to about 8.5. This latex antibody conjugate was dialyzed (Spectra-por dialysis tubing, molecular weight cutoff of 300,000, Spectrum, Houston, Tex.) against 4 changes of 2 L each of 20 mM sodium borate/150 mM sodium chloride, pH 8.2 at 4.degree. C. for 4 days. The dialyzed latex conjugate was then removed from the dialysis tubing and the solids concentration was calculated to be 0.4%. This conjugate can be used for immunoassays for hCG in serum. The latex has excitation and emission wavelengths of 650 nm and 780 nm, respectively.
A solution of polystyrene sulfate latex (0.036 mL, 8.4% solids, 1000 nm; Interfacial Dynamics Corp., Inc., Portland Oreg.) was added quickly, at room temperature, while vortexing to a solution consisting of anti-.alpha. hCG monoclonal antibody (0.12 mL, 10.3 mg/mL; Applied Biotech Inc. San Diego, Calif.) in 20 mM sodium borate/150 mM sodium chloride, pH 8.2 and 0.1M potassium citrate, pH 3, (0.6 mL). The solution incubated at room temperature for 5 minutes and was subjected to centrifugation in an Eppendorf centrifuge (2000.times.g for 5 min). The supernatant was removed, the pellet was resuspended in 0.1M potassium phosphate, pH 7, (1.5 mL) and the suspension was subjected to centrifugation as described above. This process was repeated 2 times more and in the final centrifugation, the pellet was resuspended with 0.1M potassium phosphate, pH 7 (0.3 mL) to make 1% solids. This antibody latex is used on a solid phase, such as a membrane, to capture the hCG-dye antibody latex conjugate complex in a reaction mixture in an immunoassay for hCG.
EXAMPLE 17
Immunoassay for hCG
The solid phase anti-a hCG latex solution (0.005 mL, 1% solids; example 16) can be applied to a 2 cm.sup.2 piece of 0.45 micron nylon membrane (Millipore Corp., Boston, Mass.) which has been treated with a 2% solution of condensed milk to lower non-specific binding interactions. This membrane can be used as the solid phase onto which is captured the hCG dye latex conjugate complex. Thus, an hCG assay can be performed by addition of dye latex conjugate (0.025 mL, example 16) to 0.1 mL samples of serum suspected of containing hCG and also to 0.1 mL serum samples containing known amounts of hCG (10, 100, 300, 500 and 1000 mIU/mL). The serum samples should be incubated about 10 minutes and then the samples are applied to the solid phase membrane containing the solid phase latex. The membrane should be placed over an absorbent so that the serum sample containing the dye latex conjugates flows through the solid phase latex spot. After the serum solution has passed through the membrane, serum (0.5 mL) not containing the dye latex conjugate is applied to the membrane to remove unbound dye latex conjugate. The latex spots on the membranes are then placed in a front surface fluorescence accessory in a fluorometer and the spot is excited at 650 nm and the fluorescence intensity of the spot on each membrane is measured at 780 nm. The fluorescence intensity as a function of the hCG concentrations of the known samples is plotted. The fluorescence intensities of the unknown hCG serum samples can be compared to the known hCG concentrations from the graph.
EXAMPLE 18
Fluorometer for Measuring Near Infrared Emitting Dyes
The dye sample (2 mL sample volume in a 10 mm.times.10 mm quartz cuvette) was excited by a diode laser (Sun Laser SL-6; 1=670.+-.10 nm, 0.95 mW) which was filtered by a low-pass cutoff filter (Corion LS700, passes wavelengths less than 700 nm). Fluorescence emission was detected at 90.degree. to the incident diode laser beam. The emitted light was collected and focused on a silicon photodiode (Melles Griot, Cat. #13DS1009) by a condenser consisting of two aspheric lenses (Melles Griot, Cat #01 LAG 119). A high-pass cutoff filter (Schott Glass RG715) in front of the Silicon photodiode blocked scattered laser light at 670 nm but passed emitted light at wavelengths larger than 715 nm. The photocurrent from the silicon photodiode was amplified and displayed by a current amplifier in nanoamps ("nA"), (Melles Griot, Cat. #13 AMP 003). In some instances, 12 nm band filters were placed in front of the silicon photodiode with center wavelengths at 730 nm, 790 nm, 850 nm, and 900 nm.
EXAMPLE 19
Synthesis of Silicon 2,3-Naphthalocyanine bis(diphenylvinylsilyl-oxide)
A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05 mmol) in dimethylformamide (0.5 mL) containing diphenylvinylchlorosilane (28 .mu.L, 0.125 mmol) and imidazole (7 mg, 0.1 mmol) was stirred under argon at room temperature for 18 hours. The reaction mixture was evaporated and the residue purified on a silica column equilibrating with hexane and eluting the product with toluene as a long green band. The toluene fraction containing the product was evaporated to afford 5 mg green solid.
EXAMPLE 20
Synthesis of Silicon 2,3-Naphthalocyanine bis(triphenylsilyloxide)
A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05 mmol) in dimethylformamide (1 mL) containing triphenylchlorosilane (37 mg, 0.125 mmol) and imidazole (7 mg, 0.1 mmol) was stirred under argon at room temperature for 18 hours. The reaction mixture was evaporated and the residue purified on a silica column equilibrating with hexane and eluting the product with toluene as a green band. The toluene fraction containing the product was evaporated to afford 2.5 mg green solid.
EXAMPLE 21
Synthesis of Silicon 2,3-Naphthalocyanine bis(dimethylmaleimidoethoxysilyloxide)
A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05 mmol) in dimethylformamide (1 mL) containing dichlorodimethylsilane (13.5 .mu.L, 0.11 mmol) and imidazole (14 mg, 0.2 mmol) was stirred under argon at room temperature for 18 hours. The reaction mixture was then treated with N-(2-hydroxyethyl)maleimide (35 mg, 0.25 mmol) and stirred for an additional 10 hours. The reaction mixture was evaporated and the residue purified on a silica column equilibrating with hexane, then toluene and eluting the product with toluene/10% isopropanol as a green band. The eluate containing the product was evaporated to afford 3.5 mg of green solid.
EXAMPLE 22
Synthesis of Silicon 2,3-Naphthalocyanine bis(dimethylsilyloxide-trans-stilbene)
A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05 mmol) in dimethylformamide (1 mL) containing dichlorodimethylsilane (13.5 .mu.L, 0.11 mmol) and imidazole (14 mg, 0.2 mmol) was stirred under argon at room temperature for 2 hours. The reaction mixture was then treated with trans-4-hydroxystilbene (49 mg, 0.25 mmol) and stirred for an additional 5 hours. The reaction mixture was evaporated and the residue purified on a silica column equilibrating with hexane and eluting the product with toluene as a long green band. The toluene fraction containing the product was evaporated to afford 4 mg green solid.
EXAMPLE 23
Synthesis of Silicon 2,3-Naphthalocyanine bis(dimethylhexylvinyl-silyloxide)
A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05 mmol) in dimethylformamide (1 mL) containing 7-oct-1-enyldimethylchlorosilane (32 .mu.L, 0.125 mmol) and imidazole (7 mg, 0.1 mmol) was stirred under argon at room temperature for 18 hours. The reaction mixture was evaporated and the residue purified on silica column equilibrating with hexane and eluting the product with toluene as a green band. The toluene fraction containing the product was evaporated and the residue treated with hexane to afford a dark green solid and light green supernatant. The mixture was centrifuged, the supernatant removed and the solid treated with more hexane and centrifuged. The supernatant was again removed and the solid dried under vacuum to yield 7.3 mg of product.
EXAMPLE 24
Synthesis of Silicon 2,3-Naphthalocyanine bis(tridecafluoro-1,1,-2,2-tetrahydrooctyl-1-dimethylsilyloxide)
A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05 mmol) in dimethylformamide (1 mL) containing (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchlorosilane (37 .mu.L, 0.1 mmol) and imidazole (7 mg, 0.1 mmol) was stirred under argon at room temperature for 2 hours. The reaction mixture was evaporated and the residue purified on a silica column equilibrating with hexane and eluting with hexane/20% toluene followed by hexane/40% toluene to afford the product as a green band. The product eluate was evaporated and the residue treated with hexane to afford a green solid. The mixture was centrifuged, the supernatant removed and the solid treated with more hexane and recentrifuged. The supernatant was again removed and the green solid dried under vacuum to yield 7.5 mg of product.
EXAMPLE 25
Synthesis of Silicon 2,3-Naphthalocyanine bis(dimethylretinol)
A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05 mmol) in dimethylformamide (1 mL) containing dichlorodimethylsilane (13.5 .mu.L, 0.11 mmol) and imidazole (14 mg, 0.2 mmol) was stirred under argon at room temperature. After 20 minutes, the reaction mixture was treated with all-trans-retinol (72 mg, 0.25 mmol) and stirred for an additional 1 hour. The reaction mixture was evaporated and the residue purified on a silica column equilibrating with hexane and eluting the product with toluene as a long green band. The toluene fraction containing the product was evaporated and the residue treated with hexane to yield a dark green solid and light green supernatant. The mixture was centrifuged, the hexane removed and the solid dried under vacuum to yield 10 mg of final product.
EXAMPLE 26
Synthesis of Silicon Octaethoxy-2,3-naphthalocyanine Dichloride
4,9-Diethoxy-1,3-diiminobenz�f!isoindoline (0.6 g) was added under argon to freshly distilled quinoline (12 mL). After stirring for 10 minutes, silicon tetrachloride (4.0 mL) was added and the reaction mixture was heated at 190.degree. C. for 1 hour. The reaction mixture was cooled to room temperature, and water (120 mL) was added slowly to hydrolyze the unreacted silicon tetrachloride. The blue-black precipitate was filtered off and washed with methanol and acetone.
UV-vis (methylene chloride) (.lambda..sub.max nm)): 768, 869.
EXAMPLE 27
Synthesis of Silicon Octaethoxy-2,3-Naphthalocyanine Dihydroxide
A suspension of silicon octaethoxy-2,3-naphthalene dichloride (1.96 g) in pyridine (15 mL) containing water (15 mL) was refluxed for 18 hours. The suspension was cooled, the black precipitate filtered and washed with water (10 mL). The precipitate was dried under vacuum and weighed (1.37 g, purple powder).
UV-vis (methylene chloride) (.lambda..sub.max (nm)): 766, 867.
EXAMPLE 28
Synthesis of Silicon Octaethoxy-2,3-Naphthalocyanine bis(dimethylhexylvinylsilyloxide)
A suspension of silicon octaethoxy-2,3-naphthalene dihydroxide (1.0 g) in dimethylformamide (20 mL) containing 7-oct-1-enyldimethylchlorosilane (0.6 mL) and imidazole (140 mg) was stirred under argon at room temperature for 24 hours. The reaction mixture was evaporated with a rotary evaporator, chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-toluene(1:1)) vacuum dried, and weighed (46 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.-1)): 855, 370000.
Infrared Spectrum(KBr): 3074, 2958, 2924, 2854, 1589, 1417, 1373, 1348, 1262, 1238, 1194, 1161, 1111, 1044, 1025, 933, 909, 844, 799, 760 cm.sup.-1.
.sup.1 H-NMR (500 MHz, CDCl.sub.3): .delta.9.0 (m, 2,5-Nc), 7.9 (m, 3,4-Nc), 5.3 (m, --CH.sub.2), 4.6 (m, vinyl --CH.sub.2), 3.5 (m, vinyl CH), 1.8 (m, --CH.sub.3), 1.3 (m, .epsilon. --CH.sub.2 ), 0.5 (m, .delta. --CH.sub.2), 0.1 (m, .gamma. --CH.sub.2), -0.8 (m, .beta. --CH.sub.2), -1.7 (m, .alpha. --CH.sub.2), -2.3 (s, --CH.sub.3).
EXAMPLE 29
Synthesis of Silicon Phthalocyanine bis(dimethylmaleimidofluorescein)
Fluorescein ATP (0.5 mg, 1.05 .mu.mol) was treated with a solution of 0.12M potassium carbonate in 80% methanol (52 .mu.L). After 5 minutes, the hydrolysis solution was quenched by the addition of 0.5M potassium phosphate/0.1M potassium borate, pH 7.0 in 1N HCl (10 .mu.L). The quenched hydrolysis solution was evaporated to dryness, redissolved in dimethylformamide (100 .mu.L) and the resulting solution added to silicon phthalocyanine bis(dimethylmaleimidosilyloxide) in a 1.0 mL serum vial. The reaction mixture was then stirred at room temperature for 1 hour. The crude product was then chromatographed on two 3".times.3" silica plates using toluene/20% dimethylformamide. After elution, the plates were dried under vacuum and rechromatographed for a better separation. The product band was scraped off, and treated with dimethylformamide (5 mL), vortexed 30 seconds and filtered from the silica. The filtrates were evaporated to give 0.55 mg of greenish fluorescent solid.
EXAMPLE 30
Synthesis of Tin(IV) Octabutoxy-2,3-naphthalocyanine bis(triethylsilyloxide)
A mixture of triethylsilanol (77 .mu.L), sodium (3.5 mg), and xylenes (5 mL) was refluxed under argon for 1 hour and slightly cooled. A solution of tin(IV) octabutoxy-2,3-naphthalocyanine dichloride (74 mg) in xylenes (5 mL) was added to the solution formed and the mixture was refluxed for 20 minutes. The resultant was washed twice with water (25 mL each time), dried (MgSO.sub.4), and evaporated to a dark red solid with a rotary evaporator. This solid was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, toluene-isopropanol), vacuum dried, and weighed (17 mg).
UV-vis(tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.-1)): 900, 174000.
EXAMPLE 31
Synthesis of Tin(IV) 2,3-Naphthalocyanine bis(triethylsilyloxide)
A mixture of triethylsilanol (77 .mu.L), sodium (3.5 mg), and xylenes (8 mL) was refluxed under argon for 1 hour and slightly cooled. Tin(IV) 2,3-naphthalocyanine dichloride (45 mg) was added to the solution formed, and the mixture was refluxed for 5 days. The suspension was filtered, and the solid was washed (xylenes and water), vacuum dried, and weighed (41 mg). The solid was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, methylene chloride-tetrahydrofuran), vacuum dried, and weighed (26 mg).
UV-vis(tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.-1)): 700, 746; 786, 253000.
Fluorescence (tetrahydrofuran) (.lambda.max (nm)): 820.
EXAMPLE 32
Synthesis of Tin(IV) 2,3-Naphthalocyanine bis(dimethylhexylvinylsilyloxide)
A mixture of 7-oct-1-enyl dimethylsilanol (186 mg), sodium (7 mg), and xylenes (10 mL) was refluxed under argon for 4 hours and slightly cooled. Tin(IV) 2,3-naphthalocyanine dichloride (90 mg) was added to the solution formed and the mixture was refluxed for 4 days. The suspension was filtered and the solid was washed with xylenes (5 mL) and water (5 mL). The organic layer of the filtrate was separated, dried (MgSO.sub.4), and evaporated with a rotary evaporator. The residue was triturated twice with hexane (2 mL each time) to afford a bright green solid which was vacuum dried and weighed (8.5 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.1)): 670, 7200; 732, 69900; 786, 84900.
EXAMPLE 33
Synthesis of Tin (IV) Octabutoxy-2,3-naphthalocyanine Dichloride
Tin tetrachloride (234 .mu.L) was added to a mixture of octabutoxy-2,3-naphthalocyanine (310 mg) in dry dimethylformamide (15 mL) under an argon atmosphere and the mixture refluxed with stirring for 6 hours. The resultant was allowed to cool, the suspension was filtered, and the dark red solid was washed with dimethylformamide (5 mL) and water (5 mL), vacuum dried and weighed (288 mg).
EXAMPLE 34
Synthesis of Tin(IV) Octabutoxy-2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide)
A mixture of 7-oct-1-enyl dimethylsilanol (186 mg), sodium (7 mg), and xylenes (10 mL) was refluxed under argon for 5 hours and slightly cooled. Tin(IV) octabutoxy-2,3-naphthalocyanine dichloride (37 mg) was added to the solution formed, and the mixture was refluxed for 2 days. The resultant was washed with water (10 mL), dried (MgSO.sub.4), and evaporated to a dark red solid with a rotary evaporator. This solid was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, toluene-isopropanol), vacuum dried, and weighed (17 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.-1)): 785; 893, 227000.
Fluorescence(tetrahydrofuran) (.lambda..sub.max (nm)): 789.
EXAMPLE 35
Synthesis of 7-Oct-1-enyl Dimethylsilanol
A solution of 7-oct-1-enyl dimethylchlorosilane (2.56 mL) in ether (2 mL) was added dropwise over 1 hour to a stirring mixture of triethylamine (1.5 mL), water (0.18 mL) and ether (15 mL) in an ice/water bath. The resultant was stirred a further 1 hour in the ice/water bath and filtered washing the filtered solid with ether (10 mL). The filtrate was evaporated with a rotary evaporator and the residue partitioned between hexane (30 mL) and water (30 mL). The organic layer was separated, dried (MgSO.sub.4) and filtered through silica gel (70-230 mesh, 60 .ANG.), washing with hexane (100 mL). The filtrate was evaporated with a rotary evaporator to afford a colorless oil which was vacuum dried and weighed (1.06 g).
EXAMPLE 36
Synthesis of Tetrabromotetrabutoxy-2,3-naphthalocyanine
1,4-dibutoxynaphthalene-2,3-dicarbonitrile (161 mg) and 2,3-dibromo-6,7-dicyanonaphthalene (168 mg) were added to a refluxing solution of lithium metal (35 mg) in 1-butanol (2 mL) under an argon atmosphere. The reaction solution was maintained at reflux for 2 hours, cooled, and stirred into glacial acetic acid (10 mL). After 30 minutes, the solvent was evaporated with a rotary evaporator and the residue dissolved in methylene chloride (10 mL). The solution was washed twice with 1N hydrochloric acid (10 mL each time), water (10 mL), dried (MgSO.sub.4) and evaporated with a rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-toluene), the solid product triturated with hexane (2 mL), vacuum dried, and weighed (8 mg).
UV-vis(tetrahydrofuran) (.lambda..sub.max (nm)) 743; 839.
Fluorescence(tetrahydrofuran) (.lambda..sub.max (nm)): 789.
EXAMPLE 37
Synthesis of Di(1,6-dibutoxy-2,3-naphthalocyanine) di(tetrafluorophthalocyanine)
1,4-Dibutoxynaphthalene-2,3-dicarbonitrile (161 mg) and tetrafluorophthalonitrile (100 mg) were added to a refluxing solution of lithium metal (35 mg) in 1-butanol (2 mL) under an argon atmosphere. The reaction solution was maintained at reflux for 1 hour, cooled, and stirred into glacial acetic acid (10 mL). After 30 minutes the solvent was evaporated with a rotary evaporator and the residue dissolved in methylene chloride (10 mL). The solution was washed twice with 1N hydrochloric acid (10 mL each time), water (10 mL), dried (MgSO.sub.4) and evaporated with a rotary evaporator. The residue was chromatographed twice (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-toluene), the bright green fraction vacuum dried and weighed (10 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.-1)): 679, 25800; 752, 88200; 789, 76500.
Fluorescence (tetrahydrofuran) (.lambda..sub.max (nm)): 815.
EXAMPLE 38
Synthesis of Di(1,6-diphenyl-2,3-naphthalocyanine) di(tetrafluorophthalocyanine)
1,4-diphenylnaphthalene-2,3-dicarbonitrile (165 mg) and tetrafluorophthalonitrile (100 mg) were added to a refluxing solution of lithium metal (35 mg) in 1-butanol (2 mL) under an argon atmosphere. The reaction solution was maintained at reflux for 1.5 hours, cooled, and stirred into glacial acetic acid (10 mL). After 30 minutes, the solvent was evaporated with a rotary evaporator and the residue dissolved in methylene chloride (10 mL) The solution was washed twice with 1N hydrochloric acid (10 mL each time), water (10 mL), dried (MgSO.sub.4), and evaporated with a rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-toluene), the bright green fraction vacuum dried and weighed (7 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.-1)): 747, 86800.
Fluorescence(tetrahydrofuran) (.lambda..sub.max (nm)): 760.
EXAMPLE 39
Synthesis of Dibutoxy-1,3-diiminobenz�f!isoindoline
Anhydrous ammonia was slowly bubbled through a stirred mixture of 1,4-dibutoxynaphthalene-2,3-dicarbonitrile (1.61 g), 25% sodium methoxide in methanol (1.14 mL), and dry 1-butanol (10 mL) for 30 minutes. With continued ammonia introduction, the mixture was refluxed for 30 minutes. After the resultant had cooled, the solvent was removed under vacuum with a rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-toluene-isopropanol) and the yellow product treated with ether (10 mL), collected by filtration, washed with ether (10 mL), vacuum dried and weighed (517 mg).
.sup.1 H-NMR (500 MHz, CDCl.sub.3) .delta.8.22 (m, 5,8 --H), 7.65 (m, 6,7 --H), 4.23 (m, .gamma. --CH.sub.2), 1.97 (m, .beta. --CH.sub.2), 1.61 (m, .alpha. --CH.sub.2), 1.04 (t, --CH.sub.3).
EXAMPLE 40
Synthesis of Diethoxy-1,3-diiminobenz�f!isoindoline
Anhydrous ammonia was slowly bubbled through a stirred mixture of 1,4-diethoxynaphthalene-2,3-dicarbonitrile (1.33 g), 25% sodium methoxide in methanol (1.14 mL), and dry ethanol (10 mL) for 20 minutes. With continued ammonia introduction, the mixture was refluxed for 2 hours. After the resultant had cooled, the solvent was removed under vacuum with a rotary evaporator. The residue was treated with methylene chloride (10 mL) and the product was collected by filtration, washed with water (5 mL), methylene chloride (5 mL), vacuum dried and weighed (766 mg).
EXAMPLE 41
Synthesis of Silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! Diphthalocyanine Dihydroxide
Silicon tetrachloride (231 .mu.L) was added to a mixture of diphenyl-1,3-diiminobenz�f!isoindoline (470 mg) and 1,3-diiminoisoindoline (97 mg) in freshly distilled quinoline (5 mL) under an argon atmosphere and the mixture heated with stirring at 200.degree. C. for 40 minutes. The resultant was allowed to cool slightly, treated with water (5 mL) and refluxed for 5 minutes. The mixture was cooled, treated with ether (30 mL) and filtered washing the solid with ether (10 mL) and water (10 mL). The organic layer of the filtrate (which was dark green) was separated, washed with water (15 mL), dried (MgSO.sub.4) and evaporated with a rotary evaporator. The residue was chromatographed three times (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-methylene chloride), vacuum dried and weighed (55.5 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.-1)): 640; 680; 714, 67900; 742.
Fluorescence (tetrahydrofuran) (.lambda..sub.max (nm)): 750.
EXAMPLE 42
Synthesis of Silicon �di(1,6-diethoxy-2,3-naphthalocyanine)! Diphthalocyanine Dihydroxide
Silicon tetrachloride (137 .mu.L) was added to a mixture of diethoxy-1,3-diiminobenz�f!isoindoline (227 mg) and 1,3-diiminoisoindoline (58 mg) in freshly distilled quinoline (3 mL) under an argon atmosphere and the mixture heated with stirring at 200 .degree. C. for two hours. The resultant was allowed to cool slightly, treated with water (3 mL) and refluxed for 5 minutes. The mixture was cooled, treated with ether (10 mL), and the dark blue solid product filtered off, washed with ether (10 mL) and water (10 mL), vacuum dried and weighed (175 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm)): 600, 632, 666, 700, 724, 788.
EXAMPLE 43
Synthesis of Silicon �di(1,6-diethoxy-2,3-naphthalocyanine)! Diphthalocyanine bis(dimethylhexylvinylsilyloxide)
A mixture of silicon �di(1,6-diethoxy-2,3-naphthalocyanine)! diphthalocyanine dihydroxide (85 mg), 7-oct-1-enyl dimethylchlorosilane (256 .mu.L), imidazole (68 mg), and dimethylformamide (2 mL) was stirred at room temperature for 24 hours. The resultant was concentrated under vacuum with a rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-toluene-isopropanol), vacuum dried, and weighed (32 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm)): 601, 633, 667, 702, 731, 822, 904.
EXAMPLE 44
Synthesis of Silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! Diphthalocyanine bis(dimethylhexylvinylsilyloxide) (FIG. 9).
A mixture of silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! diphthalocyanine dihydroxide (30 mg), 7-oct-1-enyl dimethylchlorosilane (115 .mu.L), imidazole (30 mg) and dimethylformamide (650 .mu.L) was stirred at room temperature for 30 minutes. The resultant was concentrated under vacuum on the rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-toluene), vacuum dried and weighed (38 mg).
.sup.1 H-NMR (500 MHz, CDCl.sub.3) .delta.8.31, 8.25 (m, 2,5-Nc, 10,13-Nc), 7.94 (m, Ar--Nc), 7.95, 7.74 (3,4-Nc, 11,12-Pc), 0.68 (m, .epsilon. --CH.sub.2), 0.21 (m, .delta. --CH.sub.2), -0.11 (m, .gamma. --CH.sub.2), -1.22 (nm, .beta. --CH.sub.2), -2.14 (m, .alpha. --CH.sub.2), -2.76 (s, --CH.sub.3).
UV-vis(tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.-1)): 644; 684; 718, 81100; 748.
Fluorescence(tetrahydrofuran) (.lambda..sub.max (nm)): 754.
EXAMPLE 45
Synthesis of Tetrafluoro-1,3-diiminobenz�f!isoindoline
Anhydrous ammonia was slowly bubbled through a stirred mixture of tetrafluorophthalonitrile (2.0 g), 25% sodium methoxide in methanol (2.3 mL), and dry 1-butanol (10 mL) for 20 minutes. With continued ammonia introduction, the mixture was refluxed for 1 hour. After the resultant had cooled, the solvent was removed under vacuum with a rotary evaporator. The residue was treated with ether (50 mL) and the product was collected by filtration, washed with water (10 mL), ether (10 mL), vacuum dried and weighed (0.45 g).
EXAMPLE 46
Synthesis of Diphenyl-1,3-diiminobenz�f!isoindoline
Anhydrous ammonia was slowly bubbled through a stirred mixture of 1,4-diphenylnaphthalene-2,3-dicarbonitrile (4.3 g), 25% sodium methoxide in methanol (3.0 mL), and dry 1-butanol (25 mL) for 30 minutes. With continued ammonia introduction, the mixture was refluxed for 1.5 hours. After the resultant had cooled, the solvent was removed under vacuum with a rotary evaporator. The residue was treated with methylene chloride (50 mL) and the product was collected by filtration, washed with water (10 mL), methylene chloride (10 mL), vacuum dried and weighed (3.68 g).
EXAMPLE 47
Synthesis of Silicon �di(1.6-diphenyl-2,3-naphthalocyanine)! di(tetrafluorophthalocyanine) Dihydroxide
Silicon tetrachloride (86 .mu.L) was added to a mixture of diphenyl-1,3-diiminobenz�f!isoindoline (174 mg) and tetrafluoro-1,3-diiminoisoindoline (54 mg) in freshly distilled quinoline (1 mL) under an argon atmosphere and the mixture heated with stirring at 200.degree. C. for 1 hour. The resultant was allowed to cool slightly, treated with water (1 mL) and refluxed for 5 minutes. The mixture was cooled, treated with ether (10 mL) and filtered washing the solid with water (2 mL) and ether (5 mL). The organic layer of the filtrate was separated, washed with water (5 mL), dried (MgSO.sub.4) and evaporated with a rotary evaporator.
The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, methylene chloride), vacuum dried and weighed (18 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.-)): 727, 759, 809, 835.
Fluorescence (tetrahydrofuran) (.lambda..sub.max (nm)): 685, 760, 840.
EXAMPLE 48
Synthesis of Silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! (1,6-diethoxyphthalocyanine) Phthalocyanine Dihydroxide
Silicon tetrachloride (172 .mu.L) was added to a mixture of diphenyl-1,3-diiminobenz�f!isoindoline (347 mg), diethoxy-1,3-diiminobenz�f!isoindoline (71 mg) and 1,3-diiminoisoindoline (36 mg) in freshly distilled quinoline (2 mL) under an argon atmosphere and the mixture heated with stirring at 200 .degree. C. for 1 hour. The resultant was allowed to cool slightly, treated with water (2 mL) and refluxed for 5 minutes. The mixture was cooled, treated with ether (10 mL) and filtered washing the solid with water (5 mL) and ether (5 mL). The organic layer of the filtrate was separated, washed with water (10 mL), dried (MgSO.sub.4) and evaporated with a rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, methylene chloride), vacuum dried and weighed (6 mg).
UV-vis (methylene chloride) (.lambda..sub.max (nm)): 649, 693, 724, 758, 827.
Fluorescence (tetrahydrofuran) (.lambda..sub.max (nm)): 750.
EXAMPLE 49
Synthesis of Silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! (tetrafluorophthalocyanine) Phthalocyanine Dihydroxide
Silicon tetrachloride (172 .mu.L) was added to a mixture of diphenyl-1,3-diiminobenz�f!isoindoline (347 mg), tetrafluoro-1,3-diiminobenz�f!isoindoline (54 mg) and 1,3-diiminoisoindoline (36 mg) in freshly distilled quinoline (2 mL) under an argon atmosphere and the mixture heated with stirring at 200.degree. C. for 1 hour. The resultant was allowed to cool slightly, treated with water (2 mL) and refluxed for 5 minutes. The mixture was cooled, treated with ether (10 mL) and filtered washing the solid with water (5 mL) and ether (5 mL). The organic layer of the filtrate was separated, washed with water (10 mL), dried (MgSO.sub.4) and evaporated with a rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, methylene chloride), vacuum dried and weighed (21 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm)): 646, 689, 720, 753, 790.
Fluorescence (tetrahydrofuran) (.lambda..sub.max (nm)): 760.
EXAMPLE 50
Synthesis of Silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! (tetrafluorophthalocyanine) Phthalocyanine bis (dimethylhexylvinylsilyloxide)
A mixture of silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! (tetrafluorophthalocyanine) phthalocyanine dihydroxide (10.5 mg), 7-oct-1-enyl dimethylchlorosilane (38 .mu.L), imidazole (10 mg) and dimethylformamide (200 .mu.L) was stirred at room temperature for 30 minutes. The resultant was concentrated under vacuum on the rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-toluene), vacuum dried and weighed (4 mg).
UV-vis(tetrahydrofuran) (.lambda..sub.max (nm)): 732, 757, 794, 816.
Fluorescence(tetrahydrofuran) (.lambda..sub.max (nm)): 763, 830.
EXAMPLE 51
Synthesis of Silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! (tetrafluorophthalocyanine) Phthalocyanine bis (dimethylpentafluorophenylsilyloxide)
A mixture of silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! (tetrafluorophthalocyanine) phthalocyanine dihydroxide (10.5 mg), chlorodimethylpentafluorophenylsilane (28 .mu.L), imidazole (10 mg) and dimethylformamide (200 .mu.L) was stirred at room temperature for 30 minutes. The resultant was concentrated under vacuum on the rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-toluene) to afford two product fractions A and B which were vacuum dried and weighed (2.8 mg and 5.5 mg, respectively).
A. UV-vis(tetrahydrofuran) (.lambda..sub.max (nm)): 650, 726, 762, 796, 824.
Fluorescence(tetrahydrofuran) (.lambda..sub.max (nm)): 770.
B. UV-vis(tetrahydrofuran) (.lambda..sub.max (nm)): 651, 726, 763, 796, 824.
Fluorescence(tetrahydrofuran) (.lambda..sub.max (nm)): 770.
EXAMPLE 52
Synthesis of Silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! Diphthalocyanine bis(dimethylpentafluorophenylsilyloxide)
A mixture of silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! diphthalocyanine dihydroxide (20 mg), chlorodimethylpenta-fluorophenylsilane (58 .mu.L), imidazole (20 mg) and dimethylformamide (450 .mu.L) was stirred at room temperature for 1 hour. The resultant was concentrated under vacuum on the rotary evaporator. The residue was treated with hexane (5 mL) and the green solid product collected by filtration, washed with hexane (2 mL), vacuum dried and weighed (26 mg).
UV-vis(tetrahydrofuran) (.lambda..sub.max (nm)): 648, 691, 724, 759.
Fluorescence(tetrahydrofuran) (.lambda..sub.max (nm)): 768.
EXAMPLE 53
Synthesis of Di(1,6-diphenylnapthalocyanine)di(tert-butylphthalocyanine)
A mixture of 1,4-diphenylnaphthalene dicarbonitrile (495 mg), 4-tert-butylphthalonitrile (92 mg), and lithium butoxide (4.0 mL) was refluxed in an oil bath for 1.5 hours and cooled. Cold glacial acetic acid (20 mL) was added to the suspension formed and vacuum dried. The green residue was resuspended in dichloromethane and the solution centrifuged at 3000 rpm for 15 minutes. The supernatant was washed with 1N HCL (2.times.20mL) followed by water (1.times.10 mL). The organic layer was dried under vacuum. The crude product was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane-toluene), vacuum dried, and weighed (4.2 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm), .epsilon.(M.sup.-1 cm.sup.-1)): 668, 43297; 688, 86914; 726, 92715; 758, 64329.
Fluorescence (tetrahydrofuran) (.lambda..sub.max (nm)): 732.
EXAMPLE 54
Synthesis of 5-tert-butyl-1,3-diiminoisolindoline
Anhydrous ammonia was slowly bubbled through a stirred mixture of 4-tert-butylphthalonitrile (1.8 g), 25% sodium methoxide in methanol (2.3 mL), and dry 1-pentanol (20 mL) for 30 minutes. With continued ammonia introduction, the mixture was refluxed for 1.5 hours. After the resultant had cooled, the solvent was removed with a rotary evaporator. The residue was treated with methylene chloride (20 mL) and the product was collected by filtration, washed twice with methylene chloride (10 mL each time), ether (10 mL), vacuum dried and weighed (0.4 g).
EXAMPLE 55
Synthesis of 6,7-Dibromo-1,3-diiminobenz�f!isoindoline
Anhydrous ammonia was slowly bubbled through a stirred mixture of 6,7-dibromonaphthalene-2,3-dicarbonitrile (0.5 g), 25% sodium methoxide in methanol (0.3 mL), and dry 1-pentanol (10 mL) for 50 minutes. With continued ammonia introduction, the mixture was refluxed for 2.5 hours. After the resultant had cooled, the orange-yellow solid was collected by filtration and washed with ether (20 mL), vacuum dried and weighed (0.6 g).
EXAMPLE 56
Synthesis of Silicon �di(1,6-diphenyl-2,3-naphthalocyanine) Di-tert-butylphthalocyanine! Dihydroxide
Silicon tetrachloride (57 .mu.L) was added to a mixture of diphenyl-1,3-diiminobenz�f!isoindoline (172 mg) and 5-tert-butyl-1,3-diiminoisoindoline (50 mg) in freshly distilled quinoline (1 mL) under an argon atmosphere and the mixture heated with stirring at 210.degree. C. for 1 hour. The resultant was allowed to cool slightly, treated with water (2 mL) and refluxed for 5 minutes. The mixture was cooled, treated with ether (10 mL) and filtered washing the solid with ether (30 mL). The organic layer of the filtrate was separated, washed twice with water (20 mL each time), dried (Na.sub.2 SO.sub.4) and the ether evaporated with a rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, methylene chloride), vacuum dried and weighed (11 mg, green solid).
UV-vis (methylene chloride) (.lambda..sub.max (nm)) 656, 670, 694, 730, 758.
Fluorescence (methylene chloride) (.lambda..sub.max (nm)): 767.
EXAMPLE 57
Synthesis of Silicon �di(1,6-diphenyl-2,3-naphthalocyanine)! Di-tert-butylphthalocyanine bis(dimethylhexylvinylsilyloxide)
A mixture of silicon �di(1,6-diphenyl-2,3-naphthalocyanine) ! di(tert-butylphthalocyanine) dihydroxide (320 mg), 7-oct-1-enyl dimethylchlorosilane (200 .mu.L), imidazole (136 mg) and dimethylformamide (6 mL) was stirred at room temperature for 12 hours. The resultant was concentrated under vacuum on the rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane). The blue fraction was collected, the solvent evaporated with a rotary evaporator, and weighed (150 mg).
UV-vis (methylene chloride) (.lambda..sub.max (nm)): 632, 676, 702, 750.
Fluorescence (methylene chloride) (.lambda..sub.max (nm)): 716.
EXAMPLE 58
Synthesis of Silicon Octabromo-2,3-naphthalocyanine Dihydroxide
Silicon tetrachloride (114 .mu.L) was added to a mixture of 6,7-dibromo-1,3-diiminobenz�f!isoindoline (433 mg) and 5-tert-butyl-1,3-diiminoisoindoline (100 mg) in freshly distilled quinoline (2 mL) under an argon atmosphere and the mixture heated with stirring at 210.degree. C. for 2 hours. The resultant was allowed to cool slightly, treated with water (2 mL) and refluxed for 15 minutes. The mixture was cooled, treated with ether (4 mL) and filtered washing the solid twice with ether (2 mL each time). The solid was vacuum dried and weighed (0.57 g, dark green solid).
EXAMPLE 59
Synthesis of Silicon Octabromo-2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide)
A mixture of silicon �octabromo-2,3-naphthalocyanine dihydroxide (500 mg), 7-oct-1-enyl dimethylchlorosilane (256 .mu.L), imidazole (68 mg) and dimethylformamide (5 mL) was stirred at room temperature for 12 hours. The resultant was concentrated under vacuum with a rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, hexane), the blue-green fraction collected, vacuum dried, and weighed (300 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm)): 694.
Fluorescence (tetrahydrofuran) (.lambda..sub.max (nm)): 706.
EXAMPLE 60
Synthesis of Silicon Octaethoxyphthalocyanine Dichloride
Silicon tetrachloride (600 .mu.L) was added to a mixture of 4,7-diethoxy-1,3-diiminoisoindoline (1.0 g) in freshly distilled quinoline (10 mL) under an argon atmosphere and the mixture heated with stirring at 200.degree. C. for 1.5 hours. The resultant was allowed to cool and treated with water (10 mL) and methylene chloride (10 mL). The organic layer was separated and evaporated with a rotary evaporator. The black residue was treated with ether (5 mL) and filtered. The filtrate was dried (Na.sub.2 SO.sub.4) and the solvent evaporated with a rotary evaporator, vacuum dried and weighed(300 mg, dark green solid).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm)): 742.
UV-vis (methylene chloride) (.lambda..sub.max (nm)): 764.
Infrared spectrum (KBr): 3435, 3060, 2983, 2932, 2228, 1727, 1603, 1504, 1317, 1256, 1218, 1068, 810 cm.sup.-1.
EXAMPLE 61
Synthesis of Diethoxy-1,3-diiminoisoindoline
Anhydrous ammonia was slowly bubbled through a stirred mixture of 1,4-diethoxy-2,3-phthalonitrile (1.0 g), 25% sodium methoxide in methanol (1.2 mL), and dry 1-pentanol (20 mL) for 45 minutes. With continued ammonia introduction, the mixture was refluxed for 3 hours. After the resultant had cooled, the solvent was removed with a rotary evaporator. The residue was dried under vacuum and weighed (1.4 g, green solid).
EXAMPLE 62
Synthesis of Octamethoxy-2,3-naphthalocyanine
1,4-dimethoxynaphthalene-2,3-dicarbonitrile (820 mg) suspended in 25% sodium methoxide in methanol (7 mL) was refluxed for 1.5 hours, cooled, and stirred into glacial acetic acid (50 mL). After 30 minutes, the solvent was evaporated with a rotary evaporator and the residue dissolved in methylene chloride (100 mL). The solution was washed with 10% hydrochloric acid (100 mL), brine (100 mL) and evaporated with a rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, toluene), vacuum dried and weighed (52 mg, red-brown solid).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm)): 837.
EXAMPLE 63
Synthesis of Germanium tetra-tert-Butylphthalocyanine Dichloride
Germanium tetrachloride (1.5 mL) was added to a mixture of 5-tert-butyl-1,3-diiminoisoindoline (500 mg) and tributylamine(3.4 mL) in 1,2,3,4-tetrahydronaphthalene (7 mL) under an argon atmosphere and the mixture refluxed for 3.5 hours. The resultant was allowed to cool, treated with water (20 mL) and methylene chloride (20 mL). The organic layer was separated, washed with water (10 mL), dried (MgSO.sub.4) and evaporated with a rotary evaporator. The residue was chromatographed (silica gel 70-230 mesh, 60 .ANG., 2.times.50 cm, toluene:isopropanol (9:1)), the green fraction collected, vacuum dried, and weighed (310 mg).
UV-vis (tetrahydrofuran) (.lambda..sub.max (nm)) 680.
Fluorescence(tetrahydrofuran) (.lambda..sub.max (nm)): 718, 750.
EXAMPLE 64
Effect of Human Serum on the Fluorescence Intensities of Various Dye Systems in Latex with Different Stokes Shifts and Excitation and Emission Wavelengths
Donor and acceptor dye pairs or a hybrid phthalocyanine derivative as listed in Table 3 were incorporated into 0.2 micron latex (CML from IDC, Portland, Oreg.) using the tetrahydrofuran solvent method. The latex particles were diluted to various solids concentrations as indicated in the Table into either a buffer containing 50 mM potassium phosphate, 10 mM potassium borate, 150 mM sodium chloride and 10 mg/mL bovine serum albumin, pH 7 or neat human serum. The excitation and emission wavelengths and the corresponding Stokes shift are as indicated in the Table.
The results show that the fluorescence intensities measured in neat human serum are greatly affected when the excitation wavelength is in a region where human serum absorbs. Conversely, the fluorescence intensities of latex measured in human serum are not affected when the excitation wavelength is in the region where the serum does not significantly absorb.
TABLE 3__________________________________________________________________________ Fluores- LatexDye System Excitation Emission Stokes cence Solids(Donor/Acceptor) (nm) (nm) Shift Intensity* (%)__________________________________________________________________________trans-4-�4-(Dibutylamino) 475 680 205styryl!-1-methyl pyridiniumIodide/Silicon phthalo-cyanine bis(dimethylvinyl-silyloxide)Buffer 369.0 0.0019Serum 28.0 0.0019Meso-tetra-2-aminophenyl 420 680 260porphine/Silicon phthalo-cyanine bis(dimethylvinyl-silyloxide)Buffer 257.0 0.0010Serum 72.0 0.0010(E,E)-3,5-bis-(4-phenyl-1,3- 670 780 110butadienyl)-4,4-difluoro-4-bora-3a,4a-diazo-s-indacene/Silicon 2,3-naphthalocyanine bis(dimethylhexylvinylsilyl-oxide)Buffer 20.6 0.0005Serum 19.5 0.00051,1'-Dihexyl-3,3,3',3'- 650 780 130tetramethylindodicarbo-cyanine Iodide/Silicon 2,3-naphthalocyanine bis(dimethylhexylvinylsilyl-oxide)Buffer 28.9 0.0005Serum 30.2 0.0005Hybrid CompoundSilicon �di(1,6-diphenyl- 646 760 114naphthalocyanine)!diphthalocyanine bis(dimethylhexylvinylsilyl-oxide)Buffer 49.7 0.0007Serum 45.3 0.0007__________________________________________________________________________ *The fluorescence intensities are not corrected.
EXAMPLE 65
Effect of Axial Ligand on the Quenching of Silicon �di(1,6-diphenylnaphthalocyanine)!diphthalocyanines
Silicon �di(1,6-diphenylnaphthalocyanine)! diphthalocyanine dihydroxide and Silicon �di(1,6-diphenylnaphthalocyanine)! diphthalocyanine bis �dimethylhexylvinylsilyloxide! were incorporated into 0.2 micron CML latex (IDC Corporation, Portland Oreg.) at various dye concentrations as indicated in the Table below using the THF solvent system. The fluorescent latexes were diluted to 0.00057% solids in either 5 mM potassium phosphate, 1 mM potassium borate buffer, pH 7 or in tetrahydrofuran. The fluorescence intensities were measured by excitation at 646 nm. Emission was set at 760 nm. The results are presented below in Table 4.
The results show that the dihydroxy hybrid derivative, which has no axial ligand, has a large degree of quenching, even at 0.1 mg/mL dye loading while the bis dimethylhexylvinylsilyloxide hybrid derivative (with the axial ligand) has very little quenching. The results indicate that axial ligands are important for hybrid phthalocyanine derivatives to attain maximum fluorescence intensities in particles.
TABLE 4__________________________________________________________________________ Fluorescence Percent Quench Percent Intensity of of Silicon Fluorescence Quench of Latex con- �di(1,6- Intensity of Latex Silicon taining Silicon diphenyl- containing Silicon �di(1,6- �di(1,6- naphthalo- �di(1,6-diphenyl- diphenyl- diphenyl- cyanine)! naphthalo- naphthalo- naphthalo- diphthalo- cyanine)!Concentration cyanine)! cyanine)! cyanine bis diphthalo-of dye per mL diphthalo- diphthalo- �dimethyl- cyanine bisof 2% solid cyanine cyanine hexylvinylsilyl- �dimethylhexyl-(mg) dihydroxide dihydroxide oxidel vinylsilyloxide!__________________________________________________________________________0.1 89 1 0 40.2 75 2 6 70.3 80 2 0 100.4 78 3 2 130.6 82 2 3 160.8 84 1 5 19__________________________________________________________________________
EXAMPLE 66
Comparison of Quenching in Latex for a Hybrid Phthalocyanine Derivative and a Naphthalocyanine Derivative, Both with Axial Ligands
Silicon �di(1,6-diphenylnaphthaolcyanine)! diphthalocyanine bis�dimethylhexylvinylsilyloxide! (hybrid phthalocyanine derivative) and silicon 2,3-naphthalocyanine bis �dimethylhexylvinylsilyloxide! (naphthalocyanine derivative) were incorporated into 0.2 micron CML latex (IDC Corporation, Portland Oreg.) at various dye concentrations as indicated in the Table below using the tetrahydrofuran solvent system. The fluorescent latexes were diluted to 0.00057% solids in either 5 mM potassium phosphate, 1 mM potassium borate buffer, pH 7 or in tetrahydrofuran. The fluorescence intensities were measured at excitation and emission wavelengths as indicated in the Table below.
The results show that the hybrid phthalocyanine derivative is much more resistant to quenching than the naphthalocyanine derivative. The results show the special properties of the hybrid phthalocyanine derivatives for attaining improved fluorescence intensities in latex.
TABLE 5______________________________________Silicon 2,3- Percent Percentnaphthalocyanine Fluorescence Quench Fluorescence Quenchbis(dimethylhexyl- Intensity of (Ex. 350 Intensity of (Ex. 650vinylsilyloxide) Latex nm Latex nmconcentration (Ex. 350 nm Em. 780 (Ex. 650 nm Em 780mg/mL Em. 780 nm) nm) Em. 780 nm) nm)______________________________________0.1 11 0 1 150.3 34 13 3 300.5 41 19 4 340.7 63 26 6 410.9 31 32 3 461.0 31 28 3 422.0 33 36 3 47______________________________________Silicon �di(1,6-diphenylnaphtha-locyanine)! Percent Percentdiphthalocyanine Fluorescence Quench Fluorescence Quenchbis�dimethyl- Intensity of (Ex. 350 Intensity of (Ex. 650hexylvinylsilyl- Latex nm Latex nmoxide) concentra- (Ex. 350 nm Em. 760 (Ex. 650 nm Em 760tion (mg/mL) Em. 760 nm) nm) Em. 760 nm) nm)______________________________________0.1 11 0 6 00.3 31 0 16 00.5 56 0 28 00.7 60 0 30 00.9 78 0 39 01.0 82 0 41 02.0 113 0 58 13______________________________________

Number	Name	Date
3996345	Ullman et al.	Dec 1976
4166105	Hirschfield	Aug 1979
4199559	Ullman et al.	Apr 1980
4542104	Stryer et al.	Sep 1985
4666862	Chan	May 1987
4777128	Lippa	Oct 1988
5055414	Babb et al.	Oct 1991
5116989	Hale et al.	May 1992
5123731	Yoshinaga et al.	Jun 1992
5132206	Dreyer	Jul 1992
5154887	Babb	Oct 1992
5157412	Kleinschmidt et al.	Oct 1992
5194393	Hugl et al.	Mar 1993
5326692	Brinkley et al.	Jul 1994

Number	Date	Country
076695	Apr 1983	EPX
075982	Apr 1983	EPX
0407188A1	Jan 1991	EPX
US934334	Nov 1993	WOX

Fluorescence energy transfer and intramolecular energy transfer in particles using novel compounds

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Pekcan, O. et al, "Direct Energy Transfer Studies on Doped and Labelled Polymer Latex Particles" Physical Review Letters 61:641-644 (Aug. 1, 1988).
Stryer, Lubert, "Fluorescent Energy Transfer As A Spectroscopic Ruler" Ann. Rev. Biochem. 45: 819-46 (1978).
Molecular Probes Ad entitled "Novel Fluorescent Latex Microspheres--Tranfluospheres A Breakthrough in Latex Microsphere Technology (1994)".