An oil-soluble modified cyanine dye, its preparation method and its application

Abstract

This invention discloses an oil-soluble modified cyanine dye, its preparation method, and its application. By using organic anions to replace the halogen anions in the cyanine dye, the lipophilicity of the cyanine dye is greatly improved. The improved lipophilicity of the cyanine dye facilitates its entry into the interior of the fluorescent microspheres during the preparation process, increases its solubility in the oil phase, further improves the fluorescence performance and application value of the fluorescent microspheres, and thus meets the needs of multiple detection.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to an oil-soluble modified cyanine dye, its preparation method, and its application. Background Technology

[0002] Multiplex flow cytometry uses multiplex fluorescently encoded microspheres as a solid-phase carrier, on which proteins / nucleic acids are coupled and reacted with analytes. Then, relying on the principle of sheath flow, the individual microspheres sequentially pass through a detector for quantitative analysis. Multiplex flow cytometry is a highly sensitive and multi-parameter analytical method that combines the advantages of flow cytometry and fluorescence detection. It can simultaneously detect and analyze multiple biomolecules or cell surface markers, and has wide applications in immunology, cell biology, and molecular diagnostics.

[0003] Fluorescent-encoded microspheres play a crucial role in multiplex flow cytometry detection technology. Their main functions include: (1) signal carriers, where different fluorescent labels can be fixed on the surface of the microspheres to serve as signal carriers, with each microsphere representing a specific detection target; (2) multiplex detection, where multiple different biomolecules can be detected simultaneously by using microspheres with different fluorescent codes; and (3) cell sorting, where microspheres can also be used for cell sorting to distinguish different cell populations by fluorescent signals.

[0004] Fluorescent dyes play a crucial role as the fluorescent indicator signal in fluorescently encoded microspheres. Due to the wide variety of fluorescent dyes available, appropriate dyes can be selected based on the required encoding dimension and detection sensitivity. Cyanide dyes are derivatives of polymethylene dyes, which are interimide ion-intercalated alkenes with conjugated chromophores at both ends of the chromophore connected between N and N atoms. They are organic dyes; when the two nitrogen atoms and part of the polymethylene chain form a heterocyclic core, a typical cyanide dye is formed. Based on molecular structure, they are classified into linear, bridged, cyclobutenedione, and ketone types. Cyanide dyes are commonly used in fluorescently encoded microspheres due to their unique conjugated backbone structure, structural designability, high absorption coefficient, high fluorescence quantum yield, good chemical stability, and tunable spectral characteristics. By using different types of cyanide dyes or changing the concentration of a single dye, encoded microspheres with different fluorescence properties can be generated, enabling multiplex detection and analysis. Furthermore, the fluorescence signal of cyanide dyes can be detected in the infrared or near-infrared region, which is important for reducing background interference in biological samples.

[0005] Commonly used cyanine dyes, such as Cy3, Cy5, and Cy7, all contain the 'I' ion. - / Br- / Cl - The presence of halogen anions leads to poor lipophilicity of dyes, making it difficult for them to penetrate into the oil-phase polymer microspheres. This reduces their solubility in organic solvents such as dichloromethane and toluene, thus affecting the uniformity of dyeing and the stability of the microspheres. The most critical impact is that it limits the amount of dye that can be used in a single microsphere, thereby limiting the achievable coding range and detection multiplex, making it difficult to meet the requirements of multiplex detection.

[0006] Therefore, there is an urgent need to develop a method and products for modifying cyanine dyes, thereby increasing the types of microspheres encoded by cyanine dyes and the detection weight, and improving the performance and application value of cyanine dyes. Summary of the Invention

[0007] Based on this, the purpose of this application is to provide a rapid and simple method for modifying the lipophilicity of cyanine dyes and cyanine dye products modified using this method. This method replaces the halogen anions in the cyanine dye molecule with strongly electronegative lipophilic organic anions, thereby significantly improving the lipophilicity of the dye. This makes it easier for the cyanine dye to penetrate the interior of polymer microspheres, thus increasing its solubility in the oil phase by tens or even hundreds of times. This meets the requirements of commonly used equilibrium swelling methods and embedding methods for microsphere dyeing, achieving microsphere dyeing.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides the use of organic anions as oil-soluble modifiers in the preparation of oil-soluble modified cyanine dyes.

[0010] Furthermore, the general structural formula of the organic anion is shown in the following formula (Ⅰ):

[0011]

[0012] R1-R4 are each independently a C1-C4 alkyl, C6-C24 aryl, or 4-7 membered heterocyclic group, wherein the heteroatom in the heterocyclic group is N, S, O, or P;

[0013] B stands for boron.

[0014] Secondly, the present invention provides an oil-soluble modified cyanine dye, the general structural formula of which is shown in the following formula (II):

[0015]

[0016] Among them, R1-R6 are substituents;

[0017] R - The organic anion as described in claim 2;

[0018] Furthermore, the two benzene rings in formula (II) are replaced with naphthyl groups.

[0019] Thirdly, the present invention also provides a method for preparing an oil-soluble modified cyanine dye, comprising the following steps:

[0020] (1) Dispersing cyanine dyes using an organic solvent, wherein the organic solvent is one or more of dichloromethane, methanol, ethyl acetate, ethanol, and petroleum ether;

[0021] (2) Add an organic anionic salt to the cyanine dye solution from step (1) to carry out the reaction;

[0022] (3) Remove the organic solvent from the reaction product obtained in step (2);

[0023] (4) Using a developing solvent, separate the oil-soluble modified cyanine dye in the reaction product of step (3) by gel chromatography column;

[0024] Preferably, the developing agent is methanol and dichlorohexane in a volume ratio of (1-30):(70-99);

[0025] (5) Remove the developing agent to obtain high-purity oil-soluble modified cyanine dye crystals.

[0026] Furthermore, in the preparation method of the oil-soluble modified cyanine dye, the reaction temperature in step (2) is 2-35℃ and the reaction time is 5-10min.

[0027] Fourthly, the present invention also provides a multicolor fluorescent encoded microsphere, comprising latex seeds and a fluorescent dye encapsulated inside the latex seeds, wherein the fluorescent dye is the oil-soluble modified cyanine dye described above or an oil-soluble modified cyanine dye prepared by the preparation method described above.

[0028] Fifthly, the present invention also provides a method for preparing the aforementioned multicolor fluorescent encoded microspheres, comprising the following steps:

[0029] (1) Weigh out latex seeds and disperse them in pure water to prepare a seed dispersion;

[0030] (2) Weigh out the oil-soluble modified cyanine dye and dissolve it in an organic solvent, wherein the organic solvent includes one or more of dichloromethane, tetrahydrofuran or toluene;

[0031] (3) Add an oil-water co-solvent to the dye solution in step (2), wherein the oil-water co-solvent includes one or more combinations of ethanol, methanol, acetone, isopropanol, and n-butanol;

[0032] (4) Mix the dye solution from step (3) with the seed dispersion from step (1) to swell until swelling equilibrium is reached;

[0033] Preferably, the mixing and swelling time is 20 min to 1 h;

[0034] (5) Remove the organic solvent and wash to obtain multicolor fluorescent encoded microspheres.

[0035] Furthermore, in the preparation method of the multicolor fluorescent encoded microspheres, the particle size of the latex seeds is 100 nm to 25 μm, and the mass ratio of the latex seeds to the oil-soluble modified cyanine dye is 0.00001% to 20%.

[0036] Sixthly, the present invention also discloses the application of the multicolor fluorescent coded microspheres described above or the multicolor fluorescent coded microspheres prepared by the preparation method described above in the preparation of multiplex detection reagents.

[0037] In a seventh aspect, the present invention also discloses a diagnostic kit comprising the multicolor fluorescently encoded microspheres described above or multicolor fluorescently encoded microspheres prepared by the preparation method described above.

[0038] Compared with the prior art, this application has the following beneficial effects:

[0039] This invention provides an oil-soluble modified cyanine dye, its preparation method, and its application. By using organic anions to replace the halogen anions in the cyanine dye, the lipophilicity of the cyanine dye is greatly improved. The improved lipophilicity of the cyanine dye facilitates its entry into the interior of the fluorescent microspheres during the preparation process, increases its solubility in the oil phase, and further enhances the fluorescence performance and application value of the fluorescent microspheres. Detailed Implementation

[0040] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by way of specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of this application; however, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] The present application is described in detail below with reference to specific embodiments, which are used for understanding and not for limiting the present application.

[0044] As used herein, the term "multicolor fluorescently encoded microspheres" refers to tiny spheres made of materials such as polymers or silicone that are encoded with multiple fluorescent dyes.

[0045] As used in this article, the term "oil-water cosolvent" refers to a solvent that can dissolve both the oil phase and the water phase simultaneously.

[0046] As used herein, the term "latex seed" refers to blank polymer microspheres that can be used for staining. "Latex seeds" can be prepared using the methods described in this invention or using conventional methods in the art.

[0047] In a first aspect, the present invention provides the use of organic anions as oil-soluble modifiers in the preparation of oil-soluble modified cyanine dyes.

[0048] Furthermore, the general structural formula of the organic anion is shown in the following formula (Ⅰ):

[0049]

[0050] R1-R4 are each independently a C1-C4 alkyl, C6-C24 aryl, or 4-7 membered heterocyclic group, wherein the heteroatom in the heterocyclic group is N, S, O, or P;

[0051] B stands for boron.

[0052] Secondly, the present invention provides an oil-soluble modified cyanine dye, the general structural formula of which is shown in the following formula (II):

[0053]

[0054] Where R1-R6 are substituents,

[0055] R - The organic anion as described in claim 2;

[0056] Furthermore, the two benzene rings in formula (II) are replaced with naphthyl groups.

[0057] The cyanine dye structure of Formula (II) before organic anion substitution is derived from commercially available cyanine dyes. The general structure of Formula (II) is only used as a structural example. The R1-R6 substituents are selected based on the types and structures of commercially available cyanine dyes. The required cyanine dye can be selected according to actual detection needs.

[0058] Thirdly, the present invention also provides a method for preparing an oil-soluble modified cyanine dye, comprising the following steps:

[0059] (1) Dispersing cyanine dyes using an organic solvent, wherein the organic solvent is one or more of dichloromethane, methanol, ethyl acetate, ethanol, and petroleum ether;

[0060] (2) Add an organic anionic salt to the cyanine dye solution from step (1) to carry out the reaction;

[0061] Among them, organic anionic salts can be sodium salts, potassium salts or ammonium salts.

[0062] Among them, the reaction mode between cyanine dye and organic anion can be any method that allows the two to react fully, such as stirring, ultrasound or microwave.

[0063] (3) Remove the organic solvent from the reaction product obtained in step (2); the method of removing the organic solvent from the reaction product can be any method that can remove the organic solvent without affecting the target product, such as heating or rotary evaporation.

[0064] (4) Using a developing solvent, separate the oil-soluble modified cyanine dye in the reaction product of step (3) by gel chromatography column;

[0065] In some embodiments, the developing agent is methanol and dichlorohexane in a volume ratio of (1-30):(70-99);

[0066] (5) Remove the developing agent to obtain high-purity oil-soluble modified cyanine dye crystals; the method for removing the developing agent can be a commonly used method in this field, such as vacuum drying.

[0067] In some embodiments, the reaction temperature in step (2) of the method for preparing the oil-soluble modified cyanine dye is 2-35°C, the molar ratio of the organic anion to the cyanine dye is (1-5):1, and the reaction time is 5-10 min.

[0068] Fourthly, the present invention also provides a multicolor fluorescent encoded microsphere, comprising latex seeds and a fluorescent dye encapsulated inside the latex seeds, wherein the fluorescent dye is the oil-soluble modified cyanine dye described above or an oil-soluble modified cyanine dye prepared by the preparation method described above.

[0069] Fifthly, the present invention also provides a method for preparing the aforementioned multicolor fluorescent encoded microspheres, comprising the following steps:

[0070] (1) Weigh out latex seeds and disperse them in pure water to prepare a seed dispersion;

[0071] (2) Weigh out the oil-soluble modified cyanine dye and dissolve it in an organic solvent, wherein the organic solvent includes one or more of dichloromethane, tetrahydrofuran or toluene;

[0072] (3) Add an oil-water co-solvent to the dye solution in step (2), wherein the oil-water co-solvent includes one or more combinations of ethanol, methanol, acetone, isopropanol, and n-butanol;

[0073] (4) Mix the dye solution from step (3) with the seed dispersion from step (1) to swell until swelling equilibrium is reached;

[0074] In some embodiments, the mixing and swelling time is 20 min to 1 h;

[0075] (5) Remove the organic solvent to obtain fluorescent microspheres;

[0076] (6) Clean the fluorescent microspheres and adjust the solid content to prepare a multicolor fluorescent coded microsphere solution. The reagents used to clean the fluorescent microspheres can be ethanol, water, or a mixture of ethanol and water, or other commonly used reagents disclosed in the art.

[0077] In some embodiments, in the preparation method of the multicolor fluorescent encoded microspheres, the particle size of the latex seeds is 100nm-25μm, and the mass ratio of the latex seeds to the oil-soluble modified cyanine dye is 0.00001-20%. The specific amount can be adjusted according to the fluorescence intensity of the encoded microspheres required.

[0078] The preparation method of the multicolor fluorescent encoded microspheres described in this invention is the "water-push swelling method" (my own name). The principle is as follows: an oil-water co-solvent is used to mix the oil-soluble modified fluorescent dye dissolved in the organic solvent with the latex seeds dispersed in pure water for swelling and dyeing. After the microspheres have swollen to equilibrium, the organic swelling agent is evaporated and removed. At the same time, pure water is added to reduce the solubility of the dye in the system, thereby pushing the dye into the interior of the latex microspheres in the oil phase and improving the dyeing efficiency.

[0079] Sixthly, the present invention also provides the application of the multicolor fluorescent coded microspheres described above or the multicolor fluorescent coded microspheres prepared by the preparation method described above in the preparation of multiplex detection reagents; in the present invention, multiplex detection includes multiplex detection technologies known in the art, including flow cytometry detection, chemiluminescence detection, etc.

[0080] In a seventh aspect, the present invention also provides a diagnostic kit comprising the multicolor fluorescently encoded microspheres described above or multicolor fluorescently encoded microspheres prepared by the preparation method described above.

[0081] The present invention will be further described below through specific embodiments. Unless otherwise specified, the materials and reagents used in the following embodiments are all commonly used materials or reagents in the art, and can be obtained commercially or synthesized by known methods. Experimental methods in the following embodiments that do not specify conditions are generally carried out according to conventional experimental conditions or methods.

[0082] Example 1: Oil-soluble modification of trimethylcyanine dye

[0083] (1) Weigh 0.6g of Cy3 fluorescent dye and add it to a 100mL glass three-necked flask. Add 50mL of dichloromethane.

[0084] (2) Weigh 0.45g of sodium tetraethylborate and add it to the three-necked flask mentioned above;

[0085] (3) Ultrasonic oscillation for 5 min;

[0086] (4) Remove solvent by rotary evaporation at 40℃;

[0087] (5) Use 10 mL of dichlorohexane:methanol = 70:30 as the developing solvent to disperse the precipitated product;

[0088] (6) Using the above developing solvent, the colored substance at the foremost point is separated by silica gel chromatography column;

[0089] (7) Place the product solution in a vacuum drying oven and vacuum dry for 12 hours to collect the product; the final yield is about 97%.

[0090] Example 2: Oil-soluble modification of pentamethrin dye

[0091] (1) Weigh 0.649g of Cy5 fluorescent dye and add it to a 100mL glass three-necked flask. Add 50mL of dichloromethane.

[0092] (2) Weigh 0.45g of sodium tetraethylborate and add it to the three-necked flask mentioned above;

[0093] (3) Ultrasonic oscillation for 5 min;

[0094] (4) Remove solvent by rotary evaporation at 40℃;

[0095] (5) Use 10 mL of dichlorohexane:methanol = 70:30 as the developing solvent to dissolve and precipitate the product;

[0096] (6) Using the above developing solvent, the colored substance at the foremost point is separated by silica gel chromatography column;

[0097] (7) Place the product solution in a vacuum drying oven and vacuum dry for 12 hours to collect the product; the final yield is about 94%.

[0098] Example 3: Oil-soluble modification of pentamethrin dye

[0099] (1) Weigh 0.649g of Cy5 fluorescent dye and add it to a 100mL glass three-necked flask. Add 50mL of dichloromethane.

[0100] (2) Weigh 0.9g of sodium tetrakis(1-imidazolyl)borate and add it to the three-necked flask mentioned above;

[0101] (3) Ultrasonic oscillation for 5 min;

[0102] (4) Remove solvent by rotary evaporation at 40℃;

[0103] (5) Use 10 mL of dichlorohexane:methanol = 70:30 as the developing solvent to dissolve and precipitate the product;

[0104] (6) Using the above developing solvent, the colored substance at the foremost point is separated by silica gel chromatography column;

[0105] (7) Place the product solution in a vacuum drying oven and vacuum dry for 12 hours to collect the product; the final yield is about 97%.

[0106] Example 4: Oil-soluble modification of heptamethrin dye

[0107] (1) Weigh 0.677g of IR780 fluorescent dye and add it to a 100mL glass three-necked flask. Add 50mL of dichloromethane.

[0108] (2) Weigh out 0.45g of sodium tetraethylborate and add it to the three-necked flask mentioned above;

[0109] (3) Ultrasonic oscillation for 5 min;

[0110] (4) Remove solvent by rotary evaporation at 40℃;

[0111] (5) Use 10 mL of dichlorohexane:methanol = 80:20 as the developing solvent to dissolve and precipitate the product;

[0112] (6) Using the above developing solvent, the colored substance at the foremost point is separated by silica gel chromatography column;

[0113] (7) Place the product solution in a vacuum drying oven and vacuum dry for 12 hours to collect the product; the final yield is about 92%.

[0114] Example 5: Oil-soluble modification of heptamethrin dye

[0115] (1) Weigh 0.677g of IR780 fluorescent dye and add it to a 100mL glass three-necked flask. Add 50mL of dichloromethane.

[0116] (2) Weigh 0.9g of sodium tetrakis(1-imidazolyl)borate;

[0117] (3) Ultrasonic oscillation for 5 min;

[0118] (4) Remove solvent by rotary evaporation at 40℃;

[0119] (5) Use 10 mL of dichlorohexane:methanol = 80:20 as the developing solvent to dissolve and precipitate the product;

[0120] (6) Using the above developing solvent, the colored substance at the foremost point is separated by silica gel chromatography column;

[0121] (7) Place the product solution in a vacuum drying oven and vacuum dry for 12 hours to collect the product; the final yield is about 95%.

[0122] Example 6: Oil-soluble modification of heptamethrin dye

[0123] (1) Weigh 0.677g of IR780 fluorescent dye and add it to a 100mL glass three-necked flask. Add 50mL of dichloromethane.

[0124] (2) Weigh 1.02g of sodium tetra(p-tolyl)borate and add it to the three-necked flask mentioned above;

[0125] (3) Ultrasonic oscillation for 5 min;

[0126] (4) Remove solvent by rotary evaporation at 40℃;

[0127] (5) Use 10 mL of dichlorohexane:methanol = 90:10 as the developing solvent to dissolve and precipitate the product;

[0128] (6) Using the above developing solvent, the colored substance at the foremost point is separated by silica gel chromatography column;

[0129] The product solution was placed in a vacuum drying oven and dried under vacuum for 12 hours to collect the product; the final yield was approximately 70%. Example 7: Oil-soluble modification of heptamethrin dye.

[0130] (1) Weigh 0.677g of IR780 fluorescent dye and add it to a 100mL glass three-necked flask. Add 50mL of dichloromethane.

[0131] (2) Weigh 1.2g of sodium tetra(4-fluorophenyl)borate and add it to the three-necked flask mentioned above;

[0132] (3) Ultrasonic oscillation for 5 min;

[0133] (4) Remove solvent by rotary evaporation at 40℃;

[0134] (5) Use 10 mL of dichlorohexane:methanol = 90:10 as the developing solvent to dissolve and precipitate the product;

[0135] (6) Using the above developing solvent, the colored substance at the foremost point is separated by silica gel chromatography column;

[0136] (7) Place the product solution in a vacuum drying oven and vacuum dry for 12 hours to collect the product; the final yield is about 70%.

[0137] Example 8: Oil-soluble modification of heptamethrin dye

[0138] (1) Weigh 1.31g of iodide-1,1ˊ,3,3,3ˊ,3ˊ-hexamethylindole tricarbonyl cyanine (HITCI) fluorescent dye, add it to a 100mL glass three-necked flask, and add 50mL of dichloromethane;

[0139] (2) Weigh 0.9g of sodium tetrakis(1-imidazolyl)borate and add it to the three-necked flask mentioned above;

[0140] (3) Ultrasonic oscillation for 5 min;

[0141] (4) Remove solvent by rotary evaporation at 40℃;

[0142] (5) Use 10 mL of dichlorohexane:methanol = 80:20 as the developing solvent to dissolve and precipitate the product;

[0143] (6) Using the above developing solvent, the colored substance at the foremost point is separated by silica gel chromatography column;

[0144] (7) Place the product solution in a vacuum drying oven and vacuum dry for 12 hours to collect the product; the final yield is about 97%.

[0145] Example 9: Oil-soluble modification of heptamethrin dye

[0146] (1) Weigh 0.544g of 3,3'-diethylthiotricarbazine iodide (DTTCI) fluorescent dye, add it to a 100mL glass three-necked flask, and add 50mL of dichloromethane;

[0147] (2) Weigh out sodium tetra(1-imidazolyl)borate and add it to the three-necked flask above;

[0148] (3) Ultrasonic oscillation for 5 min;

[0149] (4) Remove solvent by rotary evaporation at 40℃;

[0150] (5) Use 10 mL of dichlorohexane:methanol = 80:20 as the developing solvent to dissolve and precipitate the product;

[0151] (6) Using the above developing solvent, the colored substance at the foremost point is separated by silica gel chromatography column;

[0152] (7) Place the product solution in a vacuum drying oven and vacuum dry for 12 hours to collect the product; the final yield is about 80%.

[0153] Example 10: Oil-soluble modification of trimethylcyanine dye

[0154] (1) Weigh 0.6g of Cy3 fluorescent dye and add it to a 100mL glass three-necked flask. Add 50mL of dichloromethane.

[0155] (2) Weigh out sodium tetra(1-imidazolyl)borate and add it to the three-necked flask above;

[0156] (3) Ultrasonic oscillation for 5 min;

[0157] (4) Remove solvent by rotary evaporation at 40℃;

[0158] (5) Use 10 mL of dichlorohexane:methanol = 70:30 as the developing solvent to disperse the precipitated product;

[0159] (6) Using the above developing solvent, the colored substance at the foremost point is separated by silica gel chromatography column;

[0160] The product solution was placed in a vacuum drying oven and dried under vacuum for 12 hours to collect the product; the final yield was approximately 97%.

[0161] Examples 1-10: Effect tests of oil-soluble modified fluorescent dyes:

[0162] Weigh 100 mg of the fluorescent dye before and after modification, add 1 mL of dichloromethane to each, seal and stir to dissolve for 24 hours at 25°C, centrifuge at 18000 rpm for 5 min to collect the liquid, vacuum dry, weigh and calculate the solubility of the fluorescent dye in dichloromethane.

[0163] (1) Changes in solubility of different fluorescent dyes before and after modification with tetraethylborate.

[0164] Table 1

[0165] Corresponding Implementation Examples Example 1 Example 2 Example 4 Solubility before modification (mg / mL) 0.14 0.2 0.3 Solubility after modification (mg / mL) 1 1.2 1.8 Solubility increased by a factor of 7.14 6 6

[0166] Table 1 shows that after oil-soluble modification with tetraethylborate, trimethylcyanine, pentamethylcyanine, and heptamethylcyanine, the solubility of the modified products in dichloromethane was greatly improved, proving the feasibility of using organic anions for oil-soluble modification to improve the solubility of cyanine dyes.

[0167] (2) Changes in the solubility of different fluorescent dyes before and after modification by different organic anions

[0168] Table 2

[0169]

[0170]

[0171] Table 2 shows that the solubility of Cy3, Cy5, and Cy7 (IR780) fluorescent dyes increased to varying degrees after oil-soluble modification with different organic anions. Among them, the organic anion with the best oil-soluble modification effect on Cy3, Cy5, and Cy7 (IR780) was tetrakis(1-imidazolyl)borate, whose solubility in dichloromethane increased by 17.14 times, 18 times, and 19 times, respectively.

[0172] (3) Changes in the solubility of organic anionic tetra(1-imidazolyl)borate groups on heptamethrin fluorescent dyes with different structures before and after modification.

[0173] Table 3

[0174]

[0175] Table 3 shows that the solubility of the three Cy7 fluorescent dyes was significantly improved after modification with the organic anion tetra(1-imidazolyl)borate.

[0176] The solubility test results above show that using organic anions to modify the oil solubility of cyanine dyes can improve their solubility. However, different organic anions improve the solubility of different cyanine dyes to varying degrees. Furthermore, since different cyanine dyes have different solubilities in organic solvents, the solubility of the modified cyanine dyes in organic solvents still varies.

[0177] Meanwhile, considering the yield of oil-soluble modified fluorescent dyes, the amount of organic anion used is chosen to ensure complete reaction and high yield of the fluorescent dye. Therefore, the amount of organic anion used in Examples 1-10 above is excessive, and all of them ensure complete reaction of the fluorescent dye. If the amount is less than this, the fluorescent dye cannot be fully utilized, thus reducing its usability.

[0178] Example 11 Preparation of fluorescent microspheres

[0179] a. Preparation of latex seeds

[0180] (1) Mix 25g of pure water and 25g of anhydrous ethanol evenly, then add 0.5g of PVP and stir evenly.

[0181] (2) Add 10g of purified styrene monomer and 0.2g of 2-phenylacrylic acid monomer to the solution obtained in step (1) while stirring at 500 rpm, and stir for 5 minutes.

[0182] (3) Add 0.05 g of ammonium persulfate to the reaction system of step (2) and heat to 70 °C. React for 12 hours.

[0183] (4) The product was washed several times by centrifugation with alcohol / water, and finally dispersed in pure water to obtain monodisperse latex seeds with a particle size of 5 μm.

[0184] b. Preparation of fluorescent microspheres

[0185] (1) Weigh 1g of latex seeds (mass of solid matter) and disperse them in 15mL of pure water;

[0186] (2) Weigh out the oil-soluble modified fluorescent dye and dissolve it in 1 mL of dichloromethane. The mass ratio of the latex seeds to the oil-soluble modified cyanine dye is 0.00001% to 20%. Adjust the specific amounts of both according to the amount of dye in Table 4 and the fluorescence intensity in Table 5.

[0187] (3) Add 15 mL of isopropanol to the dye solution from step (2);

[0188] (4) Mix the dye solution from step (3) with the latex seed dispersion from step (1) and let them swell for 30 minutes;

[0189] (5) Heat to 45℃ for 1 hour to remove dichloromethane;

[0190] (6) Centrifuge at 2000 rpm to remove the supernatant, and wash three times with ethanol and three times with pure water. After dispersing in pure water, adjust the solid content to 1%.

[0191] Example 12 Fluorescent Encoded Microspheres and Their Detection

[0192] Taking the APC / APC-Cy7 channel as an example, 5×5 multiplex coding detection was performed using Cy5 and IR780 fluorescent dye-encoded microspheres. Cy5 and IR780 correspond to the oil-soluble modified fluorescent dyes of Examples 3 and 7, respectively. The concentrations of the two fluorescent dyes were adjusted, and the microspheres were encoded according to Table 4.

[0193] Table 4

[0194]

[0195] Fluorescent microspheres with a solid content of 1% were diluted 100-fold and detected using a BeamCyte-1026 flow cytometer. The voltage settings were: SSC:FSC voltage 20, APC channel voltage 50, and APC-Cy7 channel voltage 50. Fluorescently coded microspheres prepared according to the steps in Example 11 and coded in Table 4 were detected. The fluorescence intensity results are shown in Table 5 below. From the fluorescence intensity in Table 5, it can be seen that using the Cy5 fluorescent dye of Example 3 and the IR780 fluorescent dye of Example 7 to code the microspheres allows for 5×5 multiplex detection in the APC / APC-Cy7 channel.

[0196] Table 5

[0197]

[0198] Example 12 shows that, due to the high signal requirement of the APC-Cy7 channel, a solubility of Cy7 dye greater than 3 mg / mL is required to achieve 5×5 multiplex coding detection. Therefore, the selection of organic anions is particularly important in oil-soluble modification to use Cy7 dye and achieve multiplex detection requirements, and the higher the solubility of Cy7 dye, the higher the detection multiplex that can be achieved. Among the several organic anions explored in this invention, tetra(1-imidazolyl)borate, tetra(p-tolyl)borate, and tetra(4-fluorophenyl)borate can effectively improve the solubility of Cy7, thus they are preferred for oil-soluble modification of Cy7 cyanine dye. Furthermore, based on the technical inspiration provided by these organic anions, compounds represented by their general structural formulas can also achieve the same modification effect. For Cy3 and Cy5 cyanine dyes, since the solubility requirements for these two cyanine dyes are relatively low in flow cytometry, the solubility of Cy3 and Cy5 cyanine dyes modified with the organic anions described in this invention can achieve the multiple detection objectives presented when they are combined with Cy7 cyanine dyes.

[0199] For other cyanine dyes such as Cy3.5, Cy5.5, and Cy7.5, based on the technical inspiration provided in this invention, the organic anions disclosed in this invention can also be used to modify them to improve their solubility. Experimental verification shows that the selection of organic anions for oil-soluble modification of Cy3.5, Cy5.5, and Cy7.5 can refer to the organic anions suitable for Cy3, Cy5, and Cy7, respectively.

[0200] The embodiments described above are merely examples of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application.

Claims

1. A multicolor fluorescent coded microsphere, characterized in that, It includes latex seeds and a fluorescent dye encapsulated inside the latex seeds, wherein the fluorescent dye is an oil-soluble modified anthocyanin dye. The oil-soluble modified cyanine dyes are dyes in which the halogen anions of Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5 are replaced by organic anions, wherein the organic anion is tetra(1-imidazolyl)borate. The multicolor fluorescent encoded microspheres are prepared by the following steps: (1) Weigh out latex seeds and disperse them in pure water to prepare a seed dispersion; (2) Weigh out the oil-soluble modified cyanine dye and dissolve it in an organic solvent, wherein the organic solvent is dichloromethane; (3) Add an oil-water co-solvent to the dye solution in step (2), wherein the oil-water co-solvent is one or more of ethanol, methanol, acetone, isopropanol, and n-butanol; (4) Mix the dye solution from step (3) with the seed dispersion from step (1) to swell until swelling equilibrium is reached; (5) Remove the organic solvent and wash to obtain multicolor fluorescent coded microspheres.

2. The multicolor fluorescent coded microspheres according to claim 1, characterized in that, The method for preparing the oil-soluble modified cyanine dye includes the following steps: (1) Dispersing cyanine dyes using organic solvents, wherein the cyanine dyes are any one of Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5; (2) Add an organic anionic salt to the cyanine dye solution from step (1) to carry out the reaction; (3) Remove the organic solvent from the reaction product obtained in step (2); (4) Using a developing solvent, separate the oil-soluble modified cyanine dye in the reaction product of step (3) by gel chromatography column; (5) Remove the developing agent to obtain the oil-soluble modified cyanine dye.

3. The multicolor fluorescent coded microspheres according to claim 2, characterized in that, The reaction temperature in step (2) of the preparation method of the oil-soluble modified cyanine dye is 2-35℃, and the reaction time is 5-10min.

4. The multicolor fluorescent coded microspheres according to claim 1, characterized in that, The particle size of latex seeds is 100nm-25μm.

5. The use of the multicolor fluorescent encoded microspheres according to any one of claims 1-4 in the preparation of multiplex detection reagents.

6. A diagnostic kit, characterized in that, Including the multicolor fluorescent encoded microspheres as described in any one of claims 1-4.

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