TW200925244A - Water-soluble fluorescent material with balanced hydrophilicity and hydrophobicity - Google Patents

Abstract

An amphiphilic molecule comprises a backbone and side chains grafted to the backbone. At least three backbone units are hydrophobic and fluorescent and at least one side chain unit is hydrophilic. The weight ratio within the molecule of backbone and side chain units that are hydrophilic to those that are hydrophobic is from about 1: 4 to about 4: 1. To form fluorescent particles, a solution comprising water, an organic solvent and the amphiphilic molecule dissolved in the organic solvent is provided. The concentration of the molecule in the solution is from about 1 to about 1000 CAC, such as about 10 CAC to about 100 CAC, where CAC is the critical aggregation concentration of the amphiphilic molecule. The organic solvent is removed from the solution, thus allowing the amphiphilic molecule to form particles that have a peripheral size from about 10 nm to about 10 microns.

Description

200925244 IX. INSTRUCTIONS: [Technical Field 3 of the Invention] Field of the Invention 5 ❹ 10 15 ❹ 20 The present invention relates to a fluorescent substance, particularly a water-soluble fluorescent substance, and a method of forming the same.

t -jttr J BACKGROUND OF THE INVENTION Fluorescent particles can be used in a variety of applications. For example, fluorescent groups are used, for example, as detectors, labels, or tags in many biochemical fields, such as pharmaceutical and genetic research, cell/microbiology imaging, disease diagnosis, analyte detection, and the like. Since the aqueous environment is a general environment, it is desirable to make the fluorescent particles soluble in water. However, many fluorescent groups are insoluble in water. Furthermore, when a fluorescent group is exposed to this environment, its performance may be affected by the environment and may be unstable. Thus, the desired encapsulation of the fluorescent group by a water soluble outer layer renders the formed particles soluble in aqueous solution and isolates the internal fluorescent groups from the environment. Some conventional fluorescent particles have a hydrophobic fluorescent segment and a hydrophilic or amphiphilic segment encapsulating the hydrophobic segment. However, the disadvantages of such particles are due to the aqueous environment, and such particles tend to coalesce/precipitate or disintegrate and are unstable. Therefore, performance will decay over time. Therefore, the desired one is to improve the stability of the fluorescent particles formed from the amphiphilic work light molecules. It is known that the stability of the particles formed from the amphiphilic molecules may be affected by the balance between hydrophilicity and affinity. However, it is difficult to predict that the hydrophilicity and affinity of the specific type of amphiphilic fluorescene compound (4) are sufficiently balanced with (4) stable stability. 5 200925244 [Mingna 3 Summary of Invention Amphiphilic molecules are provided to form fluorescent particles that are stable in water. Each molecule contains a backbone and side bonds grafted to the backbone. At least three of the backbone units are hydrophobic and fluorescent, and at least one of the chain units is hydrophilic. The ratio of the hydrophilic backbone of the molecule to the weight of the side chain unit to the hydrophobic is from about 1:4 to about 4:1. To form the fluorescent particles, a solution containing water, an organic solvent, and an amphiphilic molecule dissolved in an organic solvent is provided. The concentration of this molecule in the solution is from about 1 to about 1000 CAC, such as from about 1 Torr to about 100 〇 10 CAC, wherein the CAC is the critical coalescence concentration of the amphiphilic molecule. The organic solvent is removed from the solution, thereby causing the amphiphilic molecules to form particles having a peripheral size of from about 〇 n m to about 10 microns. The granules remained stable in water for more than 6 months. Accordingly, in a first aspect of the invention, a method of forming fluorescent particles 15 is provided. The method comprises providing a solution comprising water, an organic solvent, and an amphiphilic molecule dissolved in an organic solvent; the organic solvent is removed from the solution, thereby forming the amphiphilic molecule to have an outer dimension of from about 10 nm to about 10 microns. 〇 fluorescent particles. The amphipathic knife comprises a plurality of backbone units forming a molecular backbone, and a plurality of side chains grafted to the backbone of the molecule, and each side chain is formed by at least one side chain unit. At least one side chain unit in the molecule is hydrophilic, and at least three backbone units are hydrophobic and fluorescent. The ratio of the weight of the backbone unit and the side chain unit in the molecule to the hydrophobic is from about 1:4 to about 4:1, such as from about 3:7 to about 7:3. The concentration of hydrophilic molecules in the solution is from about 1 to about 1 〇〇〇 cac, such as from about 10 to about 100 CAC, wherein 6 200925244 CAC is the critical coalescence concentration of the two parent molecules. The solution can be prepared by mixing water with the first f solution containing the organic and amphiphilic molecules. The organic solvent can be removed from the solution by evaporation. The solution may have a state of about 12 to about 12 and may be at a temperature of from about 0 to about 80 ° C, such as from about 4 to about 7 〇 5 ❹ 10 15 ❹ 20 oc. According to another aspect of the present invention, there is provided a water-soluble fluorescent particle formed according to the above method. This granule may contain a ligand with a specific affinity for the selected occlusion. The ligand may be selected from the group consisting of avidin, biotin, antibodies, antigens, and DNA, and the target may be selected from the target molecule 'cell, and the organism. According to another aspect of the present invention, there is provided a molecule comprising a plurality of backbone units forming a molecular backbone, and a plurality of side chains grafted with the molecular backbone, and each side chain is formed by at least one side chain unit . The at least one side bond unit in the molecule is hydrophilic, and at least three stem units are hydrophobic and fluorescent. The weight ratio of the hydrophilic backbone unit and the side chain unit to the hydrophobic one in the molecule is from about 1:4 to about 4:1, such as from about 3:7 to about 7.3. According to another aspect of the present invention, there is provided a water-soluble fluorescent particle comprising the molecule described in the preceding paragraph. The particles may comprise a ligand having a specific affinity for the selected target. The ligand may be selected from the group consisting of avidin, biotin, antibodies, antigens, and DNA, and the target may be selected from the group consisting of a target molecule, a cell, and an organism. For the amphiphilic molecules described in the first four paragraphs, the backbone unit may comprise a fluorene unit and the side chain may comprise polyethylene glycol. The backbone unit may comprise an arylene group, a heteroaryl group, an arylene vinylene group, a heteroarylylene vinyl group, an arylene ethylene group or a heteroarylylene group unit, or a derivative thereof. The backbone unit 7 200925244 may comprise alkyl, alkoxy, alkenyl, alkynyl, alkylalkyl, arylalkylaryl, heteroaryl, aryloxy, heteroaryloxy, alkylthioalkyl Amino, dialkylamine, arylamine, diarylamine, aryl ether, heteroaryl ether, aryl sulfide, heteroaryl sulfide, dentate, cyano, nitro, a carbonyl group, a sulfinyl group, a sulfonyl group, or a perfluoroalkyl group, or a unit substituted with an amine group containing a heteroaryl group. The backbone unit may comprise a phenylene group, a porphinyl group, a helix group, a fluorenyl group, a pyridinylene group, a dipyridyl group, an oxazolyl group, an indolocarbazole group, a benzoxazole group. a phenyl group, or an oxadiazolylene unit, or a derivative thereof. At least one backbone unit can be coupled to the vinyl or ethylene group. At least two backbone units may be coupled to each other via a single carbon bond, a propyl group, or an atom selected from the group consisting of ruthenium, s, N, Si' and p. The backbone unit may comprise a flexible base that connects the two hydrophobic and honescent backbone units. The flexible group can be hydrophilic or hydrophobic. The side chain can comprise a side chain unit. The side chain unit may comprise polyethylene glycol, polyethylene diimide, (tetra)amine, polyvinyl ketone, polyacrylic acid, 15 polyethylene glycol, polylysine, or a derivative thereof. At least one side bond may be via a single carbon bond, a single disc bond, an ether group, a thioether group, an amine group, an imido group, a sulphur group, a S group, a sulfur group, a amide group, or a quinone group. Bond with the trunk. Amphiphilic molecules can have chemical formula

To 0 such as 'G' and η 1 to 2G. 1% paternity molecule can have the chemical formula 200925244

Wherein p, q, and η are integers, η is 1 to 20, and p + q is 2 to 200, such as 4 or 10. Amphiphilic molecules can have chemical formula

© 5 wherein p, q, and η are integers, η is 1 to 20, and p+q is 2 to 200. For example, in an exemplary embodiment, η =1, and p = q =1. In another embodiment, n = 1 and p = q = 2. Amphiphilic molecules can have chemical formula

The amphiphilic molecule may be formed by forming a precursor of a stem unit, the precursor® 10 comprising a hydrophobic fluorescent group; grafting a hydrophilic group to the precursor to form a grafted precursor; and grafting grafting It is formed by the formation of amphiphilic molecules. This coupling can include coupling via a coupling reaction. This linkage may include a Suzuki coupling reaction, a Grignard coupling reaction, a Stille coupling reaction, a Heck coupling reaction, a Sologashira coupling reaction, an oxidative polymerization reaction, and a reduction. The polymerization reaction, or the polycondensation reaction, is coupled. Other aspects and features of the present invention will become apparent to those skilled in the <RTIgt; </RTI> <RTIgt; </ RTI> <RTIgt; In the formula, Figures 1 to 15 show schematic chemical reaction diagrams illustrating the &amp; path of individual specific compounds of the partial + / ^ A _ example of the present invention; Figure 16 is a diagram showing the exemplification of the embodiment of the present invention. A schematic chemical reaction diagram of a general synthetic route of a compound; Figure 17 is a schematic representation of a general synthetic route illustrating a polymer of a backbone having a levisable segment of the embodiment of the invention; Figure 2 shows the formation of different compounds (measured particle size; Figure 21 is a line drawing of the measured cell number; Figure 22 is a transmission of the glare particle formed by the sample compound of the embodiment of the invention. Electron microscopy (TEM) image; and Fig. 23 is a transmission electron microscopy (TEM) image of fluorescent particles formed by another compound of the present invention. C embodiment; DETAILED DESCRIPTION It has been found that when the ratio of the weight of the hydrophilic unit to the hydrophobic unit in the molecule is from about 1:4 to about 4:1 (such as from about 3:7 to about 7:3), the graft amphiphilicity The glory molecule can be used to form a stable micelle particle having a sufficiently balanced hydrophilicity and affinity. The graft molecule has a --meridend backbone and a side chain grafted with the stem, and each of the stem and the side chain is individually at least - a stem unit or a side bond unit is formed. As understood, a unit as used herein refers to a building block of a particular moiety 200925244, for example, if a molecule is formed by polymerization of a monomer, the unit is a monomer unit. In an exemplary embodiment of the invention, the backbone comprises at least two hydrophobic fluorescent backbone units, and at least one side comprises at least one hydrophilic side chain unit. In the next embodiment, the backbone may comprise only hydrophobic backbone units. In a different embodiment, the backbone may also comprise one or more hydrophilic backbone units. To form the fluorescent particles, a solution comprising water, an organic solvent, and an amphiphilic molecule dissolved in an organic solvent is provided. The concentration is about Up to about 1000 CAC, such as from about 10 to about 100 CAC, wherein the CAC is a critical coalescence concentration of the amphiphilic molecule. The organic solvent is removed from the solution, thereby causing the amphiphilic molecule to form with about 1 〇 nm to Micelle particles of about 1 micron in size. The particles can remain stable in water for more than 6 months. Although not limited by any particular theory, it is considered that the balance of hydrophilicity and affinity in the molecule is based on the weight ratio. It is achieved when it is within the above range. It can be understood that the balance between hydrophilicity and affinity affects the stability of the particles in water. Although the hydrophilicity of the molecule is too strong, the particles are easily disintegrated in water. When it is too strong, the particles tend to coalesce or sink in the water. In either case, the particles are unstable in water. When the hydrophilicity of the molecule and the weight ratio of the hydrophobic I1 unit are within the ranges indicated above, the interaction between the various segments within the particle and the aqueous environment is in dynamic equilibrium. Therefore, the particles 20 remain stable in water or in an aqueous environment. The particles are considered to be stable when their dimensions are the same over a long period of time (such as - weeks or up to 6 months). The change in particle size from size is considered to be substantially the same for a particular application in which the particles are used. Dimensional variations may be allowed for different applications. 11 200925244 The stability of the granules in aqueous solution can be determined using any suitable technique. For example, the solution can be visually detected over time to see if it remains clear. If the particles are precipitated or coalesced, a precipitate or a solution that is not clear will be observed. If the solution is kept clear, the size of the solution can be measured or monitored by the silk technology (such as ', 5 light scattering technology). The size of the glory is measured, for example, ζ·(4) et al. 'Langmuir, _, the first boots = page; and W. Brown et al, j. Phys. Chem, 1991, page 95, the first coffee page; each The content is hereby incorporated by reference. In the illustrated embodiment, the grafting molecule can be a co-light polymer or a co-pressing cold 10 polymer. The backbone is formed from a backbone unit comprising at least three hydrophobic and fluorescing units. The unit of hydrophobicity and fluorescing can be a repeating unit. The side chain contains hydrophilic side chain units. If no other hydrophobic or hydrophilic segments are present in the molecule, the weight ratio of hydrophilic side chain units to hydrophobic repeat units in the molecule can range from about 1:4 to about 4:1. In certain embodiments, the weight ratio can range from about 3:7 to about 7:3. The backbone may comprise an arylene group, a heteroaryl group, an arylene vinylene group, a heteroarylylene group, an arylene group, or a heteroaryl group ethylene unit, or a derivative thereof. In the preceding sentence or other similar content, "or" means that each of the items listed is a possible alternative, and any combination of any two of the items listed in the item is also a possible alternative. The backbone unit may also be a stupid base, a porphinyl group, a helical diterpene group, an anthracene fluorene group, a pyridyl group, a bismuth base, a carbazole group, an indolocarbazole group, a benzoxazole group, a sulphur beta-study unit, or a derivative thereof. In one embodiment of the invention, the repeating unit in the backbone may comprise a fluorene unit. In one embodiment, the backbone unit may be 12 200925244 The vinyl group or the ethylene group is bonded. The trunk sheet may also be bonded to one or two cc triple bonds. In another embodiment, the stem unit may be via a single carbon bond, a methylene group, or selected from O, S, N, The atoms of Si and P are coupled to each other. In various embodiments, the backbone unit may comprise a single 5-membered with an organic group. The organic group may be an alkyl group, an alkoxy group, an alkenyl group, an alkynyl group, an alkylalkyl group. , arylalkylaryl, heteroaryl, aryloxy, heteroaryloxy, alkyl-thio, alkylamino, Alkylamino, arylamine, diarylamine, aryl ether, heteroaryl ether, aryl sulfide, heteroaryl sulfide, halogen, cyanide, nitro, carbonyl, sulfoxide a sulfonyl group, or a perfluoroalkyl group, or an amine group containing a heteroaryl group. The substituted group may be selected to improve the solubility of the forming molecule in a selective solvent. The solvent may be an organic solvent such as tetrahydrofuran. (THF), gas, dioxane, toluene, etc. For example, the molecule may comprise a hydrazine block substituted with a hexyl group. The backbone may further comprise a flexible 15 group linking the fluorescent units in the backbone. The group may be hydrophilic or hydrophobic. For example, the flexible group may be an alkyl group, a substituted or unsubstituted alkyl group, a dilute group, an alkyne group, an ether group, a thioether group, an amine group, An imido group, an ester group, a thioester group, a decylamino group, a guanidino group, a decyl group, etc. The substituent may contain any of the groups listed above, any substituted or unsubstituted aryl or hetero An aryl group, any hetero atom, any combination of 20 or the like. The side chain may comprise a side bond unit which thus comprises a hydrophilic The hydrophilic early element of the side bond or the backbone may comprise polyethylene glycol (peg), polyethylenediamine, polyamine, polyvinylpyrrolidone, polyacrylic acid, polyvinyl alcohol, or polylysine. Acid, or a derivative thereof. The hydrophilic unit in the side chain may be via a single 13 200925244 or a w bond, a thiol group, a thioether group, an amine group, an imido group, a calcined group, a vine group, a quinone group, a hydrazine group, a hydrazine group The amine group or the quinone imine group is bonded to the backbone. The hydrophilic side chain can be bonded to any position of the stem at the end or in the middle of the stem. If the chemical structure or chemical formula of a specific molecule is provided, the hydrophilicity and hydrophobicity within the molecule are provided. The sex unit (segment) can be easily identified by those skilled in the art. Then, the weight ratio of the unit (segment) of the two types in the knife can be calculated based on the atomic weight of the element existing in the individual block and the segment (segment). The total weight of all hydrophilic segments within the molecule and the total weight of all hydrophobic segments can be calculated individually and used to calculate the ratio. Alternatively, for a polymer or oligomer formed from repeating monomer units ❹ 10, the weight ratio of the two segments in each monomer unit of the polymer can be calculated and used to determine the weight ratio of the entire polymer. The weight ratio can also be determined by another suitable method known to those skilled in the art. In one embodiment, the grafted molecules can be formed as follows. The first of the main units is from a commercially available source (such as Sigma-AldrichTM,

FlukaTM, MerckTM, TCLTM, Alfa Aesar, prepared or obtained. Preferably, the quality can be prepared using any suitable technique known to those skilled in the art. At least 〇 Some precursors contain hydrophobic fluorescent groups. The side chain comprising a hydrophilic side chain group is grafted with the individual precursors to form a grafted precursor. The grafted precursor can be the same or a different molecule. The grafted precursor is then joined to form the final grafted molecule. The ratio of side bonds to precursors is selected such that the weight ratio of hydrophilic segments to hydrophobic segments is from about 1:4 to about 4:1, such as from about 3:7 to about 7:3. Each grafted precursor can be a monomer, a polymer, or a polymer. The stoichiometric ratio of the different grafted precursors in the grafted molecule can be varied, 14 200925244 and depending on the desired stem size, molecular weight, solubility, bioavailability, biocompatibility, optical properties, or other correlation Choose from features. The grafted precursors can be coupled together via a coupling reaction. The coupling reaction can be a Suzuki coupling reaction, a Grignard coupling reaction, or a Stiller coupling reaction. The grafted 5 precursor may also be linked to form a grafted molecule via an oxidative polymerization reaction, a reduction polymerization reaction, a polycondensation reaction, a Heck reaction, a Sologashira reaction or the like. As will be appreciated by those skilled in the art, grafted precursors can be directly coupled or indirectly coupled by other backbone units. The coupled backbone of the grafted precursor may have no side chains attached thereto or one or more side chains attached thereto. For example, the coupling stem unit can be a flexible base. In a different embodiment, the grafted molecules can be formed using other procedures. For example, the 'polymer backbone can be prepared first, and then the side chains are grafted to the backbone in a post polymerization process. In one example, a PEG having a 0H end group or an amine group can be grafted to a polymer backbone comprising an alkyl bromide group, an acid vapor group, or an anhydride group. In another example, a side chain having an acid chloride group or an anhydride group may be grafted to a backbone comprising a hydrazine H group or an amine group. The post-polymerization technique is illustrated in Cuihua ❺ Xue et al., "Facile, versatile prepolymerization and postpolymerization functionalization approaches for well-defined fluorescent conjugated fluorene-based 20 glycopolymers", Macromolecules, 2006, Vol. 39, No. 17, 5747-5752 Page; Κ· Buga et al., "Postpolymerization grafting of aniline tetramer on polythiophene chain: Structural organization of the product and its electrochemical and spectroelectrochemical properties&gt;,, Chemistry Of Materials, 15 200925244 2005, Vol. 17, No. 23,5754-5762 Page; K. Buga et al., "Poly (alkylthiophene) with pendant dianiline groups via postpolymerization functionalization: preparation, spectroscopic, and spectroelectrochemical characterization", 5 Macromolecules, 2004, Vol. 37, No. 3, 769-777; and JS

Liu and R. D. McCullough, "End group modification of regioregular polythiophene through postpolymerization functionalization", Macromolecules, 2002, vol. 35, No. 27, pp. 9882-9889, the contents of each of which are incorporated herein by reference. 10 Certain exemplary embodiments of the invention relate to water soluble fluorescent particles formed from grafting molecules as described above. The particles may have an outer dimension of from 1 〇 nm to 10 μm. When the particles are spherical, the peripheral size is the particle diameter. When the particles have an irregular shape or a non-uniform size, the peripheral size refers to the effective or average diameter of the particles. The effective diameter of the non-spherical particles is the diameter of the spherical particles of the same volume as those of the non-spherical 15 diameter. The size of the particles can be measured using any suitable technique&apos; including optical or electronic imaging techniques. For example, the size of the particles can be measured using light scattering techniques. 0 Particles may contain ligands with a specific affinity for the selected target. For example, the ligand can be avidin, biotin, antibody, antigen, or DNA, 20 and the target can be a molecule, a cell, or an organism. In one example, the particles can be formed as follows. A first solution containing a pure molecule dissolved in an organic shaft is obtained. The organic solvent may be THF, gas, dioxane, toluene or the like. The first solution T is a product solution of a self-grafting method. Depending on the circumstances 16 200925244 5 ❹ 10 15 ❹ 20 The 'product solution' can be further selected or treated as necessary. The first solution can also be prepared individually by dissolving the grafting molecule in an organic solvent. The first solution is mixed with water to form a second solution. To form the second solution, water or an aqueous solution may be added to the first solution, or the first solution may be added to water or an aqueous solution. The aqueous solution contains water and may also contain a pH buffer solution. The concentration of the grafted molecules in the second solution needs to be controlled. Assuming that the specific grafting molecule has a critical coalescence concentration in the second solution, the concentration of the grafted molecules in the second solution in the CAC needs to be from about 1 to about 1000 CAC in one embodiment, and in another embodiment About 10 to about 100 CAC. The CAC of a particular graft molecule in a particular solution can be determined by monitoring the intensity of the light emitted from the solution as the concentration of the grafted molecule is increased. When the concentration of the grafted molecules in the solution is at or near the CAC value, the luminescence intensity exhibits a sharp increase. CAC can be based on, for example, K.

Holmberg et al. 'Handbook of applied surface and colloid chemistry, 2002, Vol. 2, Chapter 13 discloses a method of measuring the measurement', the contents of which are hereby incorporated by reference. The organic solvent is secondarily removed from the second solution, thereby allowing the grafted molecules, for example, to form particles by self-assembly. The organic solvent can be removed by evaporation or another technique. For example, a rotary evaporation technique can be used. Evaporation of the organic solvent can be promoted by exposing the second solution to a vacuum, by heating, or by both. In certain embodiments, at least 9% of the organic solvent needs to be removed. The self-assembling method is motivated by the interaction of hydrophilic and hydrophobic interactions between the grafted molecules and different segments in the water. Typically, the hydrophobic segments and the water molecules repel each other, while the hydrophilic segments and water molecules attract each other. To facilitate the self-assembly method, the second solution can have from about 2 to about 12 ipH, depending on the application of 17 200925244. The second solution can be maintained at about 〇 X to about 80 during evaporation and particle formation. (The temperature, such as, from about 4 〇c to about 7〇%. 5 The forming solution and the particles formed therein may be subjected to any suitable post-forming treatment, such as extrusion, purification, drying, etc., depending on the application. In different embodiments, the granules can be formed using different procedures. For example, the granules can be formed by forming an amphiphilic pure segment of silk into a mixture solution of water and organic, and the organic solvent can be self-mixed in different ways. Extraction of the solution solution. In the method of (4) (dialysis method), the mixture solution is transferred to the dialysis tube, and then immersed in a water bath. Due to the difference in concentration, the organic solvent will diffuse from the tube into the water bath. (eg, every two (2) hours) update. The update method can be repeated until the organic solvent is removed from the solution in the tube. Typically, this method can take two to three days to complete. The remaining solution in the tube will Contains formed particles. The dialysis procedure can be used immediately when the organic solvent is not very volatile. Another solvent removal technique is the sonic injection method, which can be used when the organic solvent is volatile. For example, or acetone. In this technique, the mixed solution can be slowly injected under the sonication treatment (in the pure water accumulation. The formed solution can be sonicated for a period of time, such as about 10 minutes. The sonicated solution is Stir at room temperature, for example, for 2 days to remove the organic solvent by evaporation.

20 It is contemplated that the fluorescent particles formed by the above exemplary methods are soluble in hydrazine and in aqueous environments such as water, and (4) for a longer period of time, up to 6 months, exhibit stable glory release ( See, for example, Example I3) below. In comparison, when the nanoparticles of the nanometer size are used with similar sites: the weight ratio of the hydrophilic segment to the hydrophobic segment in the particle is formed when the amphiphilic molecule outside the range of w to 18 200925244 4:1 is formed. The particles may be unstable in water, and their performance such as fluorescence detection is expected to deteriorate in a relatively short period of time (such as from 1 day to one week). For example, an amphiphilic graft polymer 2,7-monobromo-9,9(6-polyethylene glycol-hexyl)anthracene and 2_bromo-9,9(6,_polyethylene glycol_hexyl) 5苟It has been found to be unstable in water. The weight ratio of the hydrophilic segment to the hydrophobic segment in the two compounds is about 10:1. Conversely, the test results show that when the weight ratio is in the range of about 1:4 to about 4:1, and the concentration of the grafted molecules in the second solution is from about 1 to about 1000 CAC (such as from about 10 to about 100 CAC). At the time, the test sample grafted molecules formed fluorescent particles that were observed to be stable in the aqueous phase at an extended period of time (such as more than 6 months). In a particular embodiment, the backbone of the grafted molecule comprises oligosaccharides or polyfluorenes, and the side chains comprising PEC^PEG units can have any suitable size or weight. The grafted precursor of the grafted molecule can be coupled via a Suzuki coupling reaction. The weight ratio of PEG to oligo or polyfluorene in the trowel is about 1:4 to 4:1, or about 3:7 to about 7.3. In the first solution, the organic solvent may be terpene or dihalogen methane. The first product can be added to the water by dripping when the mixed solution is stirred. The organic agent can be removed by evaporation. The formed particles may have a size ranging from several nanometers to several micrometers. The first solution and the particles formed in the water will exhibit a strong glory emission. Fluorescence can be measured using a fluorescence spectrometer or a confocal spectrometer. The intense fluorescence exhibited by such particles can be used for imaging or detection applications, for biological applications. For example, the particles can be attached to a particular ligand. The vector may be a nucleotide, a single strand of DNA, a double stranded DNA, a single strand of RNA, a biguanide-15 τντ a , a peptide, a protein, a hormone, an antibody, a receptor, an antigen, an antigen 19 200925244 determinant, a nucleic acid binding protein, Molecules, enzyme substrates or analogs thereof, avidin, streptavidin, biotin, monosaccharides, polysaccharides, and the like. Particles with specific ligands can be used to calibrate specific cells or organisms. When the particle size is generally in the nanometer range, it can be easily absorbed by cells or organisms. The particles may also be prepared to be compatible with the selected environment, such as surrounding cells. For example, the components of the granules can be selected such that the presence of the granules does not undesirably act, such as being toxic to the cells. EXAMPLES Example 1 (Synthesis of Compound 1) ® 10 Compound 1 is 2,7-dibromo~(9,9-dihexyl)anthracene prepared according to the reaction shown in Figure 1. 2,7-Dibromoindole (9.72 g, 30 mmol, obtained from Sigma-Aldrich) was added to 8 〇〇c of aqueous sodium hydroxide (54.3 ml, 50%), tetrabutylammonium bromide (1 82 Gram, 5.63 millimolar), and a mixture of 1_bromohexane (25.36 ml '180 mmol). The mixture was cooled to room temperature after 5 hours of mixing. The mixture is mixed with dioxane to extract the reaction product. The organic layer was washed sequentially with water, aqueous HC1, water, and brine. The layer washed with 0 was dried using anhydrous MgS04. The remaining solvent and excess 1-bromohexane were removed. The residue was purified by vermiculite gel column chromatography using hexane as the eluent. The purified product contained a white solid in a weight of 20 14.55 g, corresponding to a yield of 98.6% by weight. The measured spectrum of the white solid: NMR (CDC13, 400MHz) δ[ρρπι]: 7.513 (m,6H), 1.908 (m, 4H), 1.119 (m, 12H), 0.782 (t, 6H), 0.583 ( m, 4H), confirmed product containing compound 1 '2,7-di-di-(9,9-dihexyl) 3⁄4. Example 2 (Synthesis of Compound 2) 20 200925244 Compound 2 is 2,7-bis(4,4,5,5-tetramethyl-U,2-dioxaborane-2-yl) 9,9-dihexyl芴, which is prepared according to the reaction shown in Figure 2, using compound i. 2.46 g (5 mmol) of Compound 1 was added to THF (5 mL) to form a solution. The solution was cooled to -78. (: 15 ml (18 mmol) of n-butyllithium (1.2 M 'obtained from AldrichTM) was added to the solution to form a mixture. The mixture was mixed for 1 hour and the solution temperature was maintained at _78〇c. 2_Isopropoxy-4,4,5,5-tetradecyl-1,3,2-dioxaborane (2.7 ml, 12.98 mmol) was added rapidly to the mixture. Warm to room temperature, and stir for 10 overnight. The mixture is poured into water. The reaction product in the mixture is extracted with ether. The extraction method is repeated 3 times. The extracted organic layer is mixed and washed with brine and anhydrous magnesium sulfate. Drying. The remaining solvent was removed by rotary evaporation. The residue was purified by column chromatography (ethyl acetate:hexane = 1: 15). The purified product contained 1.823 g of white solids. Yield of % by weight.) The measured spectrum of the white solid is 1H NMR (CDC13, 400 MHz) 8 [ppm]: 7.793 (d, 2H, J = 7.6 Hz), 7.742 (s, 2H), 7.725 (d , 2H, J=7.6Hz), 1.995 (m, 4H), 1.386 (s, 24H), 1.064 (m, 12H), 0.740 (t, 6H), 0.546 (m, 4H), confirm the product compound 2 ' 2,7- (4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) 9,9-dihexyl 20 芴. Example 3 (Synthesis of Compound 3) Compound 3 Series 2 -Bromo-(9,9-dihexyl)anthracene, which is synthesized according to the reaction shown in Figure 3. 2-bromoindole (12.25 g, 50 mmol) is added to 8 (TC of aqueous sodium hydroxide) 91 ml, 50%), a mixture of tetrabutylammonium bromide (3.03 g, 9.38 21 200925244 mmol) and 1-bromohexane (42.3 ml, 300 mmol). After stirring for 4 hours, the mixture was cooled to After extraction with di-methane, the mixed organic layer was washed successively with water, aqueous HC1, water, and brine, and then dried in anhydrous MgSCU. The remaining solvent was removed from the dried layer and excess 5 1 After the bromohexane, the residue was purified by vermiculite gel column chromatography using hexane as the eluent. The product formed contained 20. 3 g (97 wt% yield) of pale yellow liquid product. And the measured spectrum is 4 NMR (CDC13, 400MHz) 5 [ppm]: 7.681 (m, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.45 (m, 2H), 7.35 (m, 3H) ), 1.95 (m,4H), 1.09 (m, 12H), 0.79 (t, 10 】=7.1 along, 611), 0. 62 (111, 411). The product is the compound 3,2-bromo-(9,9-dihexyl)anthracene. Example 4 (Synthesis of Compound 4) Compound 4 is 2-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)-9,9-dihexylfluorene It is prepared by using Compound 3 according to the reaction shown in Fig. 4. 62 g (15 mmol) of compound 3 was mixed with 100 ml of anhydrous THF to form a solution. 22.5 ml of n-BuLi (27 mmol) was added to this solution at -78 °C. The solution was stirred for 1 hour before adding 2_isopropoxy-4 4 5 5 -tetramethyl H2-dioxaborane (3.9 mL, 18.75 mmol). The resulting mixture was mixed overnight. Then, water is added to further the reaction. The reaction product in the mixture 20 was extracted three times with di-methane (100 ml). The extracted organic layer was washed with brine dried over anhydrous MgSO.sub.4 and then concentrated in vacuo. The concentrated layer was subjected to column chromatography (elite gel, ethyl acetate: hexane = 1 : 20) to yield a product of 5.83 g (yield: 8.45 wt.). The measured spectrum of the product is 1H NMR (CDC13, 400MHz) δ[ρριη]: 7.812 (m, 200925244 1H), 7.747 (m, 3H), 7.322 (m, 3H), 7.322 (m, 3H), 1.990 ( m, 4H), 1.39 (s, 12H), 1.023 (m, 12H), 0.752 (m, 6H), 0.597 (m, 4H), confirmed product 4, 2-(4,4,5,5- Tetramethyl-1,3,2-dioxaborane-2-yl)-9,9-dihexylfluorene. 5 Example 5 (Synthesis of Compound 5) Compound 5 is 2,7-dibromo-9,9-bis(6,-bromohexyl) 3⁄4, which is prepared according to the reaction shown in Figure 5. 2,7-Dibromoindole (0.972 g, 3 mmol) was added to aqueous potassium hydroxide (60 ml, 50%) at 75 ° C, tetrabutylammonium bromide (0.198 g, 0.6 mmol). 'And a mixture of 1,6-two-seat (7.32 g, 30 mmol). After stirring for 15 minutes, the mixture was cooled to room temperature. After extraction with methylene chloride, the combined organic layers were washed successively with water, aqueous HCl, water, and brine, and then dried over anhydrous MgS04. After removing the remaining solvent and excess 1,6-dibromohexane, 'residue was analyzed by vermiculite gel column chromatography using hexane: gas (v/v: 9:1) as eluent And purified. The product formed contained 15 white solids of 1.47 g (75 wt% yield), and the measured spectrum was 1 NMR (CDC13, 400 MHz) δ (ppm): 7.433-7.535 (m, 6H), 3.294 ❹ ( t, 4H), 1.923 (m, 4H), 1.652-1.687 (m, 4H), 1.203 (m, 4H), 1.083 (m, 4H), 0.587 (m, 4H), confirm the product compound 5, 2, 7-Dibromo-9,9-bis(6,-bromohexyl)anthracene. 20 Example 6 (Synthesis of Compound 6) Compound 6 is 2,7-dibromo-9,9(6'-polyethylene glycol-hexyl)anthracene, which is based on the reaction shown in Figure 6 using Compound 5 preparation. Sodium hydride (0.9 g, 40 mmol) and dry THF (30 mL) were placed in a three-neck 150 ml flask under argon. Then, PEG (8 g, 4 mmol) 23 200925244 in THF (50 ml) was added dropwise to /BZL. As shown in Figure 6, the molecular weight of peg used is 2000 Daltons, expressed as PEG2000 or PEG2_. However, it is understood that PEG2000 is used for testing purposes, and in various embodiments of the invention, the weight of PEG can vary. 5 The resulting mixture was stirred for 4 hours. Next, 0.65 g (1 mmol) of Compound 5 was added to the flask. The mixture in the flask was stirred at room temperature for about 1 week. In this case, water is added by dripping to terminate the further reaction. The organic solvent in the mixture was evaporated, and the residue was dissolved in 20 ml of di-methane and 4 ml of methanol. The solution was mixed with 500 ml of ether to form a precipitate. The precipitate was collected by centrifuging the solvent with ❹10. The precipitate was further purified by repeating the above method 3 times, and then dried under vacuum. The product formed is compound 6. Example 7 (Synthesis of Compound 7) Compound 7 is 2-bromo-9,9-bis(6,-bromohexyl)anthracene which is prepared according to the reaction of Figure 7. 2-bromoindole (4.9 g, 20 mmol) was added to a 75 ° C 15 aqueous potassium hydroxide (400 mL, 50%), tetrabutyl bromo bromide (1.32 g, 4 mM) and 1 a mixture of 6-dibromohexane (48.8 g, 200 mmol). After stirring for 15 minutes, the mixture was cooled to room temperature. After extraction with digas methane, the mixed organic layer was washed successively with water, aqueous hydrazine α, water, and brine, and then dried in anhydrous MgS〇4. Remove the remaining solvent and excess sputum, after the 6_two deserts have been burned, the residue is eluted by lithography/gas imitation (v:v = 9:1) by Shi Xishi gel column chromatography. Purified by liquid. The product was a pale yellow liquid, weighed 8 6 g (75 wt% yield), and the measured spectrum is iH NMR (CDCl3, 4 〇〇 MHz) δ (ppm): 7.669 (m, 1H), 7.548 (m, 1H) , 7.453 (m, 2H), 7.322 (m, 3H), 3.280 (t, 4H), 1.943 (m, 4H), 1.657 (m, 4H), 1.193 24 200925244 (m,4Η),1·073 (m , 4Η)' 0.604 (m, 4Η), confirming the product is a compound 7,2-di-9,9-bis(6'-indolyl)indole. Example 8 (Synthesis of Compound 8) 5 φ 10 15 ❹ 20 Compound 8 is a 2-bromo-9,9(6'-polyethylene glycol-hexyl) fluorene which is used according to the reaction shown in Figure 8 7 and synthesized. Sodium hydride (0.96 g &apos; 40 mmol) and anhydrous THF (3 mL) were placed in a three-neck 150 ml flask under argon. PEG (8 g '4 mmol) in thf (50 ml) was added dropwise in the chamber. The mixture was disturbed for 4 hours. Next, '0.57 g (1 mmol) of compound 7 was added to the mixture. The mixture was further stirred at room temperature for about one week. Water was then added dropwise to the flask to terminate the further reaction. The solution was evaporated to remove the THF. The remaining mixture was dissolved in 20 ml of di-methane and 4 ml of methanol, and then mixed with 500 ml of ether to form a precipitate. The solvent in the mixture is removed by centrifugation. The above precipitation method was repeated 3 times. The precipitate formed was dried under vacuum. The product formed is compound 8. Example 9 (Synthesis of Compound 9) Compound 9 was prepared according to the reaction shown in Figure 9. A mixture of 2.63 g (15 mmol), dibromide (22 mmol) and anhydrous potassium carbonate 1.38 g (10 mmol) was dissolved in acetone and heated to reflux. After refluxing for 8 hours, water was added to terminate the further reaction. The mixture was mixed with 50 ml of ether (3 times) to extract an organic precipitate. The precipitate was washed successively with 2M gas oxidized sodium solution, brine, and then dried in anhydrous MgS〇4. After removal of the remaining solvent, the crude product was recrystallized from ethanol to give compound 9' weighs 1.15 g (46 wt% yield), and the measured spectrum is ι 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 -7.347 (d, 4H, J = 8.8 Hz), 7.776-7.754 (d, 4H, J = 8.8 Hz), 3.929 (t, 4H), 1.803 (m, 4H), 1.533 (m, 4H). 13C NMR (CDC13, 400 MHz) δ (ppm): 158.429, 132.409, 116.548, 112.885, 68.299, 29.290, 25.982. 5 Example 10 (Synthesis of Compound 10) Compound 10 is represented by 3F1-PEG2000, which was prepared according to the reaction shown in Figure 10 using Compounds 2, 3 and 4. Compound 2 (0.15 g, 0.25 mmol), Compound 4 (1.103 g, 0.25 mmol), tetrakis(triphenylphosphine) palladium (50 mg, 0.04 mmol), aqueous sodium carbonate (2M, 1.24) ML), a mixture of 10 and toluene (10.16 ml) was deoxygenated and then heated to reflux under nitrogen. The mixture was stirred for 4 hours. Next, 0.103 g (0.18 mmol) of Compound 3 dissolved in 1 ml of toluene was added to the mixture. The mixture was further stirred for about 2 days and then cooled to room temperature. The organic solvent in the mixture is evaporated. The residue was dissolved in 4 ml of di-methane and mixed with 100 ml of ether to form a precipitate. The solvent is removed by centrifugation. The precipitation method was repeated 3 times. The crude product was dissolved in dioxane and subjected to dialysis for about 8 weeks using an 8k dialysis tube. The solution is then lyophilized to form a pale powder product which is the compound 1 〇. The yield of the 〇 synthesis method was 24% by weight. In compound 10, the PEG segment is hydrophilic and the other segments are hydrophobic. The weight ratio of the hydrophilicity to the hydrophobic unit in the molecule of the compound 10 is about 4: Compound 1 in water (^CAC system 〇〇 5 mg/ml. For the preparation of stable particles from the compound 1 ,, 1 〇 of the compound 1 The hydrazine was dissolved in 1 mL of water (equal to 2 〇 CAC). The resulting solution was scrambled for 3 days. The micelle granules were formed in solution and remained stable for 6 months during storage. 26 200925244 Example 11 (Compound 11 Synthesis) 5 ❹ 10 15 ❹ 20 Compound 11 is a 3F2-PEG2000 prepared according to the reaction shown in Figure 11 using Compounds 4 and 6. Compound 4 (0.46 g, 1 mmol), Compound 6 (1.123 g, a mixture of tetrakis(triphenylphosphine) (50 mg, 〇·〇 4 mmol), aqueous sodium carbonate (2M, 1.24 ml), and toluene (10.16 ml) was deoxygenated' then The mixture was heated to reflux under nitrogen, and the mixture was stirred for 2 days, then 'cooled to room temperature. The organic solvent in the mixture was evaporated. The residue was dissolved in 20 ml of dichloromethane and mixed with 800 ml of ether to form a sink. The solvent was removed by centrifugation. The precipitation method was repeated 3 times. The product bath was simmered in a wind and subjected to dialysis using an 8k dialysis tube for about 1 week. The solution was cold/east dried to form a pale powder product which was compound 11. The yield of this method was 50%. The weight ratio of the hydrophilicity to the hydrophobic segment in the compound u is about 4: 1. The weight average molecular weight (Mw) and the number average molecular weight (Mn) of the compound 11 are based on gel permeation chromatography (GPC) analysis. And pEG was determined as a standard for calibration, and individual lines 4123 and 3969 were found. The Gpc result means that not 11 molecules of each compound only contain - the block in which the pEG side chain is attached to the thiol group. The CAC of compound 11 is used. Dynamic Light Scattering (DLS) technique was applied to the aqueous solution at room temperature (IV) and found to be hunger mg/ml. The compound of CCA in water (4) was 0.10 mg/ml. For the preparation of self-chemical substance 11, a stable teaching, 1 〇 mg The compound was excreted in 1 liter of tip at 1G CAC). The shaft is reduced for 3 days. The micelle granules are formed in solution and are kept for 6 months during storage. 27 200925244 Example 12 (Synthesis of Compound 12) Compound 12 is a 5F3-PEG2000 which was prepared according to the reaction shown in Figure 12 using Compounds 2, 3 and 6. Compound 2 (0.23 g, 0.4 mmol), Compound 3 (0.25 g '0.6 mmol), Compound 6 (0.45 g, 0.1 mmol), 5 (triphenylphosphine) palladium (32 mg, 0.028) A mixture of aqueous sodium carbonate (2M, 0.88 ml) and toluene (2.6 mL) was deoxygenated and then heated to reflux under nitrogen. The mixture was refluxed for 2 days and then cooled to room temperature. The organic solvent in the mixture was evaporated and the residue was dissolved in 20 ml of methane. The solution was mixed with 800 ml of ether to form a precipitate. The solvent is removed by treatment from 10 hearts. The precipitation method was repeated 3 times. The crude product was dissolved in digas methane and subjected to dialysis using an 8 k dialysis tube for approximately one week. The solution was lyophilized to form a pale powder product which was compound 12. The yield of this example was 46%. The weight ratio of hydrophilic to hydrophobic units in compound 12 is about 2.4:1. The weight average molecular weight (mw) and number average molecular weight (?n) of the compound 12 were determined based on GPC analysis and PEG was determined as a standard for calibration, and individual lines 4409 and 3919 were found. The GPC results indicate that each compound 12 molecule contains only one block in which the PEG side chain is attached to the thiol group. The CAC of Compound 12 was measured in aqueous solution at room temperature using DLS technique and found to be 0.08 mg/ml. 20 To prepare stable granules from compound 12, 10 mg of compound 12 was dissolved in 10 ml of water (equal to 12.5 CAC). The resulting solution was stirred for 3 days. Micelle particles are formed in solution which is stable for 6 months during storage. Example 13 (Synthesis of Compound 13) Compound 13 (PF1-PEG2000) was prepared according to the reaction shown in Figure 3, using 200925244 with Compounds 1, 2 and 6. The symbols ρ and q used here represent integers. As shown in Figure 13, P+q=19. Compound 1 (〇.〇89 g, 0.18 mmol), Compound 2 (0.117 g, 0.2 mmol), Compound 6 (0.0898 g, 〇.〇2 mmol), tetrakis(triphenylphosphine) (2 mg, 0.002 mmol), a mixture of aqueous sodium carbonate 5 (2M, 0.284 ml) and toluene (1.15 ml) was deoxygenated and then heated to reflux under a gas atmosphere. The mixture was refluxed for 2 days and then cooled to the chamber. The organic solvent in the mixture was evaporated and the residue was dissolved in 4 mL of dichloromethane. The solution was mixed with 100 ml of ether to form a precipitate. The solvent is removed by centrifugation. The precipitation method was repeated 3 times. The crude product was dissolved in dioxane and subjected to dialysis for 1 week using a l〇k dialysis tube. Then, the solution was freeze-dried to form a pale yellow powder product, Compound 13. The resulting product has a particle size of about 85 nm. The yield of this example was 14%. The measured spectrum of the product is 1H NMR (CDC13, 400MHz) δ[ρριη]: 7.854 (m, 30H), 7.674 (m, 90H), 3.643 (m, 360H), 2.129 (m, 80H), 1.414 (m, 15 240H), 0.795 (m, 200H). The weight ratio of the hydrophilicity to the hydrophobic unit in the compound 13 is about 1:1.6. The CAC of Compound 13 was found to be 0.008 mg / 毫升 ml. The fluorescence and stability of Compound 13 were tested. For this purpose, BV-2 cells were cultured for 2 days. An aqueous solution containing 〇.3 mg/g of nanoparticles formed from the compound 2〇 13 (PF1-PEG2000) was prepared as follows. 3 mg of compound 13 was dissolved in 1.5 ml of THF to form a starting solution. The initial solution was further diluted with THF to a total volume of 2 ml of the forming solution. 8 ml of deionized water was slowly added to the formed solution over a period of 3 hours. The solution is contained in a bottle. The bottle was covered with a piece of KimwipesTM 29 200925244, allowing the THF to evaporate during 3 days. After 3 days, water was added to adjust the concentration of the final solution, which had a total volume of 1 mL. The final solution contained about 0.3 mg/g of compound 13 (equal to about 4 〇cac). The solution was added to the medium in a ratio of 1:1 〇 (iv). The cells were maintained in the base for 6 hours. Next, the cells were fixed with 4% polymethylation for 2 hours and then washed 3 times with PBS. The cells were observed under a co-focus microscope. Two batches: Compound 13 was used for this test. The first batch of compound (10) was newly prepared. The second batch of compound 13 was stored in aqueous solution at room temperature for at least 6 months. In the second case, the fixed cells show fluorescence of similar intensity. In another test, the cultured BV_ cells were sedimented after 2 days of culture. Next, an aqueous solution containing particles formed from the compound 13 as described above is added to the medium. The different test lines were carried out in different batches, wherein the concentration of the aqueous solution in the medium was 1%, 2%, 5%, 1%, or 2% (v/v), respectively. 15

For each batch, the cell viability of BV2 cells was measured four times at 12-hour intervals (ie, 12, 24, 48, and 72 hours after addition). Representative measurements are shown in Figure 21. . For batches in which the concentration of the particulate solution is in the range of 1% to 5%, the number of cells measured in the medium is significantly increased over time. For batches with higher concentrations (10% and 2%), the increase was less significant. This is expected when the solvent of the granule solution is water. A significant increase in the number of cells 20 indicates that the fluorescent particles of Compound 13 are non-toxic or have only very low toxicity to cells. It was observed that after the cells were activated, the cells could absorb more fluorescent particles formed from the compound 13. This suggests that the particles act as a carrier for delivering the drug or other substance to the selectively activated cells/organisms. 30 200925244 Example 14 (Synthesis of Compound η) Compound 14 is a PF2-PEG2000' which was prepared according to the reaction shown in Figure 14 using Compounds 1, 2, 6, and 9. Like the symbols "p" and "q", the symbol η also represents an integer when used herein. In Fig. 4, the value of n may be 1 to 5 1 , such as 1 to 20, and p + q = 10. Compound 1 (〇16 g, 〇·32 mmol), Compound 2 (0.28 g, 0.48 mmol), Compound 6 (0.36 g, 0.08 mmol), Compound 9 (40 mg, 〇.〇8 m Mole), tetrakis(triphenylphosphine) (78 mg '〇.〇7 mmol), aqueous sodium carbonate (2 Μ, 〇93 ml), a mixture of hydrazine and toluene (3·72 ml) was deoxygenated Then 'heated back to 10 streams under nitrogen. The mixture was refluxed for 2 days and then cooled to room temperature. The organic solvent was steamed and the residue was sown in a 10 ml broth. The solution is mixed with 400 ml shout to form a sink; The solvent is removed by centrifugation. The precipitation method was repeated 3 times. The crude product was dissolved in dioxane and subjected to dialysis using a 10 k dialysis tube. About one week. The solution was lyophilized to form a pale yellow powder product, compound 15 14 . The product particles have a size of about 154 nm. The yield was 37% by weight. The weight ratio of the hydrophilicity to the hydrophobic unit in the compound 14 is about 丨9. The CAC of the compound 14 was found to be 0.00032 mg/ml. The aqueous solution containing the particles formed from the compound 14 was prepared by the same procedure as described for the compound 13 and substituting the compound 14 for the compound 13. The final aqueous solution contained about 0.3 mg/ml of compound 14 (equal to about 940 CAC). The micelle particles in the final solution remained stable for 6 months during storage. Example 15 (Synthesis of Compound 15) Compound 15 is PF3-PEG2000, which was prepared according to the reverse 31 200925244 shown in Figure 15 using Compounds 1, 2, 6, and 9. In Fig. 15, 'n may be 1 to 100' such as 1 to 20, and p + q = 4. Compound 1 (0.16 g, 0.32 mmol), Compound 2 (0.28 g, 0.48 mmol), Compound 6 (0.36 g, 0.08 mmol), Compound 9 (40 mg, 0_08 mmol), A mixture of triphenylphosphine)palladium (64.1 5 mg, 0.055 mmol), aqueous sodium carbonate (2M, 0.88 mL), and toluene (3 mL) was prepared. The mixture was treated as described in Example 14. The product formed was a pale powder product, compound 15. The product particles have a size of about 178 nm. The yield was 21% by weight. The weight ratio of the hydrophilicity to the hydrophobic unit in the compound 15 is about 5:2. The CAC of Compound 15 found Ο 10 system 0.0004 mg/ml. The aqueous solution containing the particles formed from the compound 15 was prepared by the same procedure as described for the compound 13 and substituting the compound 15 for the compound 13. The final aqueous solution contained about 0.3 mg/ml of compound 15 (equal to about 750 CAC). The micelle particles in the final solution remained stable for 6 months at the time of storage. Example 16 BV-2 cells were cultured for 2 days and then activated by adding different concentrations of stimulating sputum, lipopolysaccharide, for 24 hours. An aqueous solution containing the fluorescent particles formed from Compound 13 was prepared as described in Example 13. The solution was added to the cell culture 20 nutrient. The volume ratio of the solution to the medium was 1:1 〇〇. Fluorescence from the medium was monitored using conjugate imaging techniques. It was observed that as the stimulation intensity increased (after the irritant), the fluorescence emission from the cells became stronger, indicating that more of the fluorescent particles had been inhaled by the activated cells. As can be appreciated, the synthetic path of the above embodiment can be modified or generally 32 200925244

Other graft molecules in accordance with various aspects of the invention are made. For example, a water-soluble fluorescent polymer can generally be formed in accordance with the reaction shown in Figure 16, and a polymer having a flexible segment in the backbone can be prepared according to the reaction shown in Figure 。. 5 The value of the reaction 'X, y, z, p, q, and η (which represents an integer) shown in Fig. 16 can be changed. The sum, p+q (=x+y+z), can also be changed. As understood, compound 13 was formed, x = 9, y = 10, z = 1; and at compound 13, 10 15 2 〇 p = 1, q = 19, and 1 ! In various embodiments, the value of p+q can vary, such as from 2 to 200. Molecules having a different sum of p+q can be prepared by adjusting the relative molar or weight ratio of the reactants present in the reaction mixture. Generally, a fluorescent polymer forming stable nanoparticles can be formed as follows. The concentration of the reactants in the appropriate amounts of the compounds 1, 2, and 6 with the tetrakis(triphenylphosphine)palladium, the aqueous sodium carbonate, and the fluorene-benzene mixed complex may vary. In the examples, the concentration of the total reactants can be q i ML 〇 Μ. The mixture can be deoxygenated and then heated to reflux under nitrogen. Mixture 4 is refluxed and (4)-stage is extended for a period of time (such as 2 days to _, after cooling, to a lower temperature (such as room temperature). The organic solvent in the mixture is treated. The residue is soluble. The minimum amount of dichloro-r or another suitable solvent. The new solution and the appropriate amount of Weichin _. New _ volume ratio can be as strong as 1: Dissolution by, such as centrifugation removal. It is repeated, for example, 3 times. The obtained crude product is soluble in the two-dimensional corrective agent, and can be refined (such as the use of his dialysis tube to extend the time of the observation, such as about 3 days to - week) Bu Rong liquid «cold green dry silk end silk, which contains the desired grafting molecule 33 200925244 into particles. The size of the particles can be controlled by changing the molecular structure, molecular weight, or solution concentration. The reaction of Figure 16 and the above procedure are prepared, wherein the concentration of the reagent is selected such that p = 1 and the total number of backbone units is 18. 5 The C AC system forming the compound is measured in an aqueous solution at room temperature using the DLS technique. And found to be 0.008 mg / ml. This sample compound The average molecular weight (Mw) and the number average molecular weight (Mn) were determined based on Gpc analysis and PEG was used as the calibration standard, and individual lines 19585 and 9886 were found. The value of Mn was also calculated based on NMR measurement. And found to be 10 9976. The GPC results indicate that the sample compound molecule contains only one block in which the PEG side chain is attached to the thiol group. To prepare the compound according to the reaction shown in Figure 17, a similar procedure is followed, but in addition The compound, compound 9, is included in the starting mixture. The symbols a, b, c, d, and r in the figure also represent integers. The molar ratio between compound 9 and other components of 15 may be 1. :100 to 1:2, such as 1:5〇 to 1:2. Generally, p can be 1 to 10, q can be m2〇〇, r(=d) can be ι2〇, and η can be 1 to 1〇〇'such as, 1 to 2〇. The values of a, b, c, and d can be selected according to the desired final product. As understood, when r = ,, the figure of Figure 17 and the 16th The compounds shown in the figure are the same. In different embodiments, the sum of 2〇 of p+q can be changed, such as 2 to 200. From the reaction path according to Figure 10 or 17 The particles formed by the prepared compound may also have a desired particle size distribution. For example, as shown in Figures 18, 19 and 20, the particle size (effective diameter) of the compound 11, 12 or 13 in the aqueous solution is from about 10 to about 300. Nm. The particle size is measured on the basis of the particle-based electron 34 200925244 microscopy (TEM). Figures 22 and 23 show an exemplary image of the fluorescent particles formed from compound 10 and compound 11 individually. A sample for sputum imaging was prepared as follows. A 9 〇pL aqueous solution of the granules was mixed with a 1 〇μί 1% aqueous PTA solution by a thirst quenching. A drop of the mixture was then placed on a 5400 mesh stainless steel grid and dried overnight with air. The particles shown in Fig. 22 have an effective diameter of 1 〇〇 to 2 〇〇 nm, and the particles shown in Fig. 23 have effective diameters ranging from 10 to 20 nm and 50 to 80 nm. Particles prepared in accordance with embodiments of the present invention as previously explored exhibit good stability in an aqueous environment. For example, the granules can remain stable in water for more than 6 months. Some of the terms used herein can be found in "Glossary of basic terms in polymer science (IUPAC Recommendations 1996)", Pure Appl. Chem., 1996, vol. 68, No. 12, pages 2287-2311. The contents are hereby incorporated by reference. 15 As is understood, embodiments of the present invention have various applications in various fields and industries. For example, the grafted molecules and particles as described above can be used in many fields. (such as biochemical field) as a fluorescent detector, label, or label. It can be used for drug and gene research, cell/microbial imaging, disease diagnosis, analyte detection, etc. 2〇 is not explicitly mentioned above. Other features, advantages, and advantages of the embodiments described herein will be apparent to those skilled in the art from the The embodiments are a number of modifications of the permissible type, part configuration, details, and sequence of operations 35 200925244. The present invention is intended to include all such improvements in the scope of the patent application. In the range of the present invention, the first embodiment of the present invention shows a schematic chemical reaction diagram illustrating the synthetic route of the embodiment of the present invention; Schematic chemical reaction diagram of the polymer route; 10 Figure 17 shows a schematic chemical reaction diagram illustrating the general synthetic route of the polymer of the backbone section of the embodiment of the present invention. Figures 18 to 20 show the formation of different compounds. Measured inch chart

Figure 21 is a line drawing of the measured number of cells; Figure 22 is a transmission electron microscopy (TEM) image of the sample compound light particles of the embodiment of the present invention; and opening &gt; Figure 23 is a transmission electron microscopy (TEM) image of fluorescent particles illustrating another sample compound of an embodiment of the present invention. [Major component symbol description] Formation (none)

36

Claims (1)

200925244 X. Patent application scope: 1. A method for forming Laoguang particles, comprising: providing a solution comprising water, an organic solvent, and an amphiphilic molecule dissolved in the organic solvent, the amphiphilic molecule comprising a plurality of forms a backbone unit of a molecular backbone 5, and a plurality of side chains grafted to the backbone of the molecule, and each of the side chains is formed by at least one side chain unit, at least one of the side chain units in the molecule being hydrophilic, And at least three of the backbone units are hydrophobic and fluorescent, and the weight ratio of the hydrophilic backbone unit and the side chain unit to the hydrophobic in the molecule is about 1:4 to about 4:1, and the solution is in the solution. The concentration of the hydrophilic 10 molecule is from about 1 to about 1000 CAC, wherein the CAC is the critical coalescence concentration of the amphiphilic molecule in the solution, and the organic solvent is removed from the solution, thereby enabling the parent The sex molecules form the particles having a peripheral dimension of from about 10 nm to about 10 microns. 2. The method of claim 1, wherein the concentration of the two 15 amphiphilic molecules in the solution is from about 10 to about 100 CAC. 3. The method of claim 1 or 2, wherein the solution is prepared by mixing water with a precursor solution comprising the organic solvent and the amphiphilic molecule. 4. The method of claim 1 or 2, wherein the removing comprises removing the organic solvent from the solution by evaporation. 5. The method of claim 1 or 2, wherein the solution has a pH of from about 2 to about 12 and is at a temperature of from about 0 to about 80 °C. 6. The method of claim 1 or 2, wherein the weight ratio is from about 3:7 to about 7:3. 37 200925244 7. The method of claim 1, wherein the backbone unit comprises a unit and the side chain comprises polyethylene glycol. 8. The method of claim 1 or 2, wherein the main unit comprises an arylene group, a hetero-aromatic base, a aryl #基乙赖基, a hetero-compound, a thief 5, a chelating group Or a heteroarylylene group unit, or a derivative thereof. - 9. The method of claim 8, wherein the backbone unit comprises - 'in the base group, alkoxy group, a dilute group, a block group, a radiant group, an aryl group, an alkyl group, a hetero group Aryl, aryloxy, heteroaryloxy, alkylthio, alkyl hydrazide, dialkylamino, arylamino, diarylamine, aryl, heteroaryl , aryl sulfonium, heteroaryl sulfide, _ cyano, cyano, nitro, # yl, sulfinyl, continuation, or decyl, or an amine substituted unit containing a heteroaryl . 10. The method of claim 1, wherein the main unit package 15 comprises a phenylene group, a fluorenyl group, a helix fluorenyl group, a cleavage group, an acridine group, a dipyridyl group, An oxazolidinyl, an indolocarbazolylbenzoxazole group, or an oxadiazolylene unit, or a derivative thereof. The method of claim 1 or 2, wherein at least one of the backbone units is coupled to a vinylene group or an ethylene group, and (2) wherein at least two of the two backbone units are via A single carbon bond, an oxime group, or an atom selected from the group consisting of ruthenium, S, N, Si, and P is bonded to each other. 12. The method of claim 2, wherein the backbone unit comprises a flexible group linking the two hydrophobic and fluorescent backbone units, the flexible substrate being hydrophilic or hydrophobic. The method of claim 1 or 2, wherein the side chain unit comprises polyethylene glycol, polyethylenediamine, polyamine, polyvinylpyrrolidone, polyacrylic acid, polyvinyl alcohol , polylysine, or a derivative thereof. 14. The method of claim 1 or 2, wherein at least one of the side chains 5 is via a single carbon bond, a monophosphorus bond, an ether group, a thioether group, an amine group, an imido group, a decyl group, an ester A thiol group, a guanidino group, or a quinone imine group is bonded to the backbone. 15. The method of claim 1 or 2, wherein the amphiphilic molecule has a chemical formula, Wherein p, q, r, and η are integers, p is 1 to 10, q is 1 to 200, r is 0 to 20, and η is 1 to 20. 16. The method of claim 15, wherein r=0. 15 17. The method of claim 1 or 2, wherein the amphiphilic molecule has a chemical formula, Wherein p, q, and η are integers, η is 1 to 20, and p+q is 2 to 200. 18. The method of claim 17, wherein p+q = 4 or 10. 19. The method of claim 1 or 2, wherein the amphiphilic molecule has a chemical formula, 39 20 200925244 20. The method of claim 19, wherein n = 1 and p = q = 1 or p = q = 2. 5. The method of claim 1 or 2, wherein the amphiphilic molecule has a chemical formula, 22. The method of claim 1 or 2, wherein the amphiphilic molecule is formed by: 10 forming a precursor of the stem unit, the precursor comprising a hydrophobic fluorescent group; The base is grafted to the precursor to form a grafted precursor; and the grafted precursor is coupled to form the amphiphilic molecule. 23. The method of claim 22, wherein the coupling comprises coupling via a coupling reaction. 15 24. The method of claim 22, wherein the coupling comprises via a Suzuki coupling reaction, a Grignard coupling reaction, a Stirling coupling reaction, a Heck coupling reaction, a Sologashira coupling reaction, an oxidative polymerization reaction, a reduction polymerization The reaction, or the coupling of a polycondensation reaction. 25. A water-soluble fluorescent particle comprising the molecule formed according to the method of the 200925244 method of claim 1. 26. A molecule comprising: a plurality of backbone units forming a molecular backbone; and a plurality of side chains grafted to the backbone of the molecule, each of the side chains being up to 5 ❹ 10 15 ❹ 20 less side chain units Forming, wherein: at least one of the side chain units in the molecule is hydrophilic, and at least three of the backbone units are hydrophobic and fluorescent, and wherein the hydrophilic backbone unit and the side chain unit pair in the molecule The weight ratio of the hydrophobic is from about 1:4 to about 4:1. 27. The numerator of claim 26, wherein the weight ratio is from about 3:7 to about 7:3. 28. The molecule of claim 26 or 27, wherein the backbone unit comprises a field unit&apos; and the side chain comprises polyethylene glycol. [29] The molecule of claim 26, wherein the backbone unit comprises an arylene group, a heteroaryl group, an arylene vinylene group, a heteroarylvinylene group, an arylethylene group Or a heteroarylylenediyl unit, or a derivative thereof. 30. The molecule of claim 29, wherein the backbone unit comprises an alkyl group, an alkoxy group, an alkenyl group, an alkynyl group, an alkyl decyl group, an aryl decyl aryl group, a heteroaryl group, an aromatic group. Oxygen, heteroaryloxy, alkylthio, alkylamino, dialkylamino, arylamine, diarylamine, aryl ether, heteroaryl ether, aryl sulfide, miscellaneous An aryl sulfide, a dentate, a cyano group, a nitro group, a carbonyl group, a sulfinyl group, a sulfonyl group, or a perfluoroalkyl group, or a unit substituted with an amine group containing a heteroaryl group. 41. The molecule of claim 26, wherein the backbone unit comprises a phenylene group, a thiophene group, a helical diamylene group, an indenoindolyl group, a pyridinylene group, a dipyridyl group. , oxazolylene, indolocarbazole, stupid and sulphate, or alpha oxa beta sulphonyl unit, or a derivative thereof. 5 32. The molecule of claim 26 or 27, wherein (1) at least one of the backbone units is coupled to an ethylene or ethylene group, and (2) wherein at least two of the backbone units are via a single carbon A bond, a methylene group, or an atom selected from the group consisting of ruthenium, S, osmium, Si, and iridium is coupled to each other. 33. The molecule of claim 26 or 27, wherein the backbone unit 10 comprises a flexible group connecting two hydrophobic and fluorescent backbone units, the flexible substrate being hydrophilic or hydrophobic. 34. The molecule of claim 26 or 27, wherein the side chain unit comprises polyethylene glycol, polyethylenediamine, polyamine, polyvinylpyrrolidone, polyacrylic acid, polyvinyl alcohol, poly Lysine, or a derivative thereof. 15 35. The molecule of claim 26 or 27, wherein at least one of the side chains is via a single carbon bond, a monophosphorus bond, an ether group, a thioether group, an amine group, an imido group, a decyl group, an ester A thiol group, a guanidino group, or a quinone imine group is bonded to the backbone. 36. The numerator of claim 26, which has a chemical formula, It is 0 to 20 ' and η is 1 to 20. 200925244 37. The numerator of claim 36, wherein r=0. 38. If the molecule of claim 26 is a chemical formula, Ο 10 PEG〇CeH12. .CeH12OPEG CgH13 CeH13 o(ch2)6o. Wherein, P, q, and n are integers, n is 1 to 20, and p+q is 2 to 200. 39. The molecule of claim 38, wherein p+q = 4 or 10. 40. If the numerator of claim 26 is of a chemical formula, pegoc6h12. .c6h12opeg CeHl3 c®Hl3 c6H13,, c6H13 wherein P, q, and n are integers, read ^ to sink, and p + q is 2 to 2 〇〇. 41. The numerator of claim 40, wherein n = 1, and p = q = 1 or P = q = 2. 42. The numerator of claim 26, which has the chemical formula, fine ~ C6H13. One, c6h12〇peg ceHi3- '〇6ηι3 43.-A kind of water-soluble (four) is difficult, including _ material range range. 26 items The particles of the 25th or 43rd patent of the patent-month patent include - a ligand with a specific affinity for the selected target. The particle of claim 44, wherein the coordination system is selected from the group consisting of a biotin, an antibody, an antigen, and a DNA, and wherein the target is a target molecule, a cell, and an organism. 44