Asymmetric cyanine compound, preparation method thereof and kit for platelet determination

Abstract

The invention provides an asymmetric cyanine compound, a preparation method thereof and a kit for platelet determination. The asymmetric cyanine compound has the following structural general formula I: # imgabs0 #, wherein Z is C (CH3) 2, O, S or Se; r1 and R2 are respectively and independently selected from H or C1-6 alkyl groups; r3 is H, a saturated alkyl group with 1 to 10 carbon atoms, an alkenyl group with 3 to 18 carbon atoms or an alkynyl group with 3 to 18 carbon atoms; r < 4 > and R < 5 > are independently selected from H, alkyl with 1 to 18 carbon atoms, OR6, alkyl with 1 to 18 carbon atoms, OR6 or halogen; r6 is a C2-18 alkyl group; and X <-> is an anion. The platelet nucleic acid dye formed by the asymmetric cyanine compound has specificity on platelet nucleic acid; under the combined action of the nucleic acid fluorescent dye liquor and the diluent, the depolymerization of the aggregated platelets can be realized, the interference of the aggregated platelets on the detection of the PLT-F channel is avoided, and the platelet aggregation samples can be accurately counted.

Description

Technical Field

[0001] The present invention relates to the technical field of biological sample staining, and particularly to an asymmetric cyanine compound, a preparation method thereof, and a kit for platelet determination. Background Art

[0002] Currently, the platelet function level is usually evaluated by the platelet aggregation rate, and the measurement can be carried out based on the principle of a flow cytometer. Currently, the fluorescence fuel solution used for detection is mostly the pigment Nile blue fluorescence fuel solution, which has the following problems: the signal-to-noise ratio of the fluorescence dye solution is low. The rigid structure of the Nile blue dye makes the fluorescence intensity of Nile blue increase less after binding to platelet nucleic acid compared with the fluorescence intensity of free Nile blue, and the fluorescence of unbound Nile blue interferes with the sample signal; interference of platelet aggregation. Currently, the supporting reagents for the platelet detection channel do not have the function of depolymerizing platelets, resulting in inaccurate platelet counting in platelet aggregation samples, and the counting fails to truly reflect the platelet function level of patients; the maximum emission peak of the free dye and the maximum emission peak of the emission spectrum after Nile blue binds to nucleic acid have a small displacement, and the fluorescence spectra overlap greatly. The fluorescence of free Nile blue easily interferes with the collection of sample signals (the fluorescence intensity of Nile blue bound to nucleic acid in platelets). To reduce the interference of the fluorescence of free Nile blue and the excitation light, a filter is commonly used, but the wavelength difference between the maximum emission peaks of free and bound Nile blue is small, and the use of the filter will also filter out a part of the fluorescence of bound Nile blue (commonly 650 nm), reducing the fluorescence use efficiency. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide an asymmetric cyanine compound, a preparation method thereof, and a kit for platelet determination in view of the defects of the prior art.

[0004] The technical solution adopted by the present invention to solve its technical problems is: an asymmetric cyanine compound having the following general structural formula I:

[0005]

[0006] Wherein:

[0007] Z is C(CH 3 ) 2 , O, S or Se;

[0008] R 1 and R 2 each independently selected from H or C 1-6 alkyl;

[0009] R3 is H, C 1-10 saturated alkyl, C 3-18 alkenyl or C 3-18 alkynyl;

[0010] R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 , -C 1-18 alkyl-OR 6 or halogen;

[0011] R 6 is C 2-18 alkyl;

[0012] X - is an anion.

[0013] Furthermore, preferably, R 1 and R 2 are each independently selected from C 2-4 alkyl;

[0014] R 4 and R 5 are each independently selected from H, C 1-4 alkyl, OR 6 , -C 5-9 alkyl-OR 6 or halogen;

[0015] R 6 is C 3-10 alkyl.

[0016] Furthermore, preferably, X - is an anion selected from a halide ion, perchlorate, hexafluorophosphate or p-toluenesulfonate, wherein the halide ion includes, but is not limited to, fluoride ion, chloride ion, bromide ion or iodide ion, etc.

[0017] Furthermore, preferably, the asymmetric cyanine compound is selected from

[0018]

[0019]

[0020]

[0021] The present invention also provides a preparation method of an asymmetric cyanine compound, comprising the following steps: S1. Reacting a compound of general formula II with an R 3 X compound to obtain a first quaternary ammonium salt intermediate III;

[0022]

[0023] S2. Reacting a 4-methylquinoline aromatic heterocyclic compound of formula IV with 1,4-dibromobutane to obtain a second quaternary ammonium salt intermediate V;

[0024]

[0025] S3. Condense the first quaternary ammonium salt intermediate III obtained in step S1 with N,N'-diphenylformamidine to obtain a compound of formula VI;

[0026]

[0027] S4. React the quaternary ammonium salt intermediate of formula V obtained in step S2 with the compound of formula VI obtained in step S3 to obtain a compound of formula VII;

[0028]

[0029] S5. React the compound of formula VII obtained in step S4 with HN(R 1 R 2 ) to obtain an asymmetric cyanine compound of formula I;

[0030]

[0031] Wherein:

[0032] Z is C(CH 3 ) 2 , O, S or Se;

[0033] R 1 and R 2 are each independently selected from H or C 1-6 alkyl;

[0034] R3 is H, C 1-10 saturated alkyl, C 3-18 alkenyl or C 3-18 alkynyl;

[0035] R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 , -C 1-18 alkyl-OR 6 or halogen;

[0036] R 6 is C 2-18 alkyl;

[0037] X - is an anion.

[0038] Furthermore, preferably in step S1, the reaction temperature is 50 - 160 °C, the reaction time is 4 - 36 hours, the reaction solvent is selected from at least one polar solvent of dichloromethane, ethanol, acetonitrile, ethyl acetate, toluene, xylene, and the compound of general formula II and R 3The molar ratio of the feed of Compound X is 1:(1 to 10);

[0039] In step S2, the reaction temperature is 50 - 160 °C, the reaction time is 4 - 36 hours, the reaction solvent is selected from at least one polar solvent of dichloromethane, ethanol, acetonitrile, ethyl acetate, toluene, xylene, and the molar ratio of the feed of Formula IV to 1,4 - dibromobutane compound is 1:(1 to 5);

[0040] In step S3, the reaction temperature is 100 - 250 °C, the reaction time is 20 minutes to 2 hours, the reaction solvent is acetic anhydride, acetic acid or a mixture thereof, and the molar ratio of the first - quaternary - ammonium - salt intermediate III to N,N’ - diphenylformamidine is 1:(1.2 to 2);

[0041] In step S4, the reaction temperature is 10 - 160 °C, the reaction time is 30 minutes - 5 hours, the reaction solvent is selected from at least one polar organic solvent of dichloromethane, chloroform, acetonitrile, ethyl acetate, methanol, ethanol, the catalyst is a mixture of acetic anhydride and an organic base, and the molar ratio of the second - quaternary - ammonium - salt intermediate of Formula V to the compound of Formula VI is 1:2 - 2:1;

[0042] In step S5, the reaction temperature is 50 - 180 °C, the reaction time is 30 minutes - 10 hours, the reaction solvent is selected from at least one organic solvent of dichloromethane, acetonitrile, methanol, ethanol and ethylene glycol monomethyl ether; the molar ratio of Compound VII to HN(R 1 R 2 ) is 1:2 - 1:30.

[0043] The present invention also provides a kit for platelet determination, which includes a diluent and a fluorescent staining solution. The pH of the diluent is 8 - 10. The diluent includes a buffer system, inorganic salts, a surfactant and a preservative. The fluorescent staining solution includes an asymmetric cyanine compound and a solvent, and the solvent is ethylene glycol.

[0044] Further, preferably, the concentration of the asymmetric cyanine compound in the fluorescent staining solution is 0.005 - 1 g / L.

[0045] Further, preferably, the buffer system is at least one of a phosphate buffer system, an ammonium chloride buffer system, and an aminoacetic acid buffer system, and preferably the concentration of the buffer system in the diluent is 0.1 g - 10 g / L.

[0046] Further, preferably, the inorganic salts are at least one of sodium chloride, sodium bicarbonate, and disodium ethylenediamine diacetate, and preferably the concentration of the inorganic salts in the diluent is 0.1 - 5 g / L.

[0047] Further, it is preferred that the surfactant is at least one of a non-ionic surfactant, an amphoteric surfactant, a positive surfactant, and a negative surfactant.

[0048] Further, it is preferred that the concentration of the preservative in the diluent is 0.5 - 4 g / L, and the preservative includes at least one of benzoic acid and its salts, isothiazolinone, sodium hydroxymethylglycinate, phenoxyethanol, and benzyl alcohol.

[0049] Advantages of the present invention: The platelet nucleic acid dye formed by the asymmetric cyanine compound of the present invention is specific to platelet nucleic acid; the wavelength difference between the maximum emission wavelength of the free dye solution formed by the asymmetric cyanine compound of the present invention and the maximum emission wavelength after the dye binds to nucleic acid is greater than 10 nm. The luminescence of the free dye solution can be filtered by a filter, reducing the overlap of the fluorescence spectra, reducing the interference of the fluorescence of the free dye solution on the sample detection (fluorescence of the bound dye), and when a filter is added to filter the emitted light and the fluorescence of the free dye, the filter will not filter out the fluorescence of the dye after binding to nucleic acid, maximizing the retention of the fluorescence intensity of the dye solution after binding to nucleic acid in cells and improving the light utilization rate. At the same time, after the dye binds to nucleic acid, its fluorescence intensity is greatly enhanced compared to the free fluorescent dye solution, improving the signal-to-noise ratio of the detection.

[0050] The nucleic acid fluorescent dye solution formed by the symmetric cyanine compound of the present invention and semiconductor laser flow cytometry can accurately count platelets in the PLT-F channel. By treating the platelet morphology with the diluent, while shaping it, the cell membrane morphology of each type is changed, facilitating the specific binding of platelet nucleic acid to the fluorescent dye solution respectively, thereby realizing the counting of platelets. In the present invention, the nucleic acid fluorescent dye solution with an amino group and the diluent act together to achieve the depolymerization of aggregated platelets, avoiding the interference of aggregated platelets on the detection of the PLT-F channel, and accurately counting platelet aggregation samples. Description of the Drawings

[0051] The present invention will be further described below in conjunction with the drawings and embodiments. In the drawings:

[0052] Figure 1 is the fluorescence spectrum diagram of dye solution A in Example 10 of the present invention and different concentrations of calf thymus DNA added to dye solution A;

[0053] Figure 2 is the fluorescence spectrum diagram of Nile blue dye solution in Example 10 and different concentrations of calf thymus DNA added to Nile blue dye solution;

[0054] Figure 3 is the emission peak wavelength difference between dye solution A in Example 10 and 10 μg / mL calf thymus DNA added to dye solution A;

[0055] Figure 4is the wavelength difference between the emission peaks of the Nile blue dye solution in Example 10 and Nile blue added with 10 ug / mL calf thymus DNA;

[0056] Figure 5 is the processing graph of platelet aggregation sample 1 by using the self-made reagent and the commercially available preparation in the PLT-F channel in Example 11;

[0057] Figure 6 is the processing graph of platelet aggregation sample 2 by using the self-made reagent and the commercially available preparation in the PLT-F channel in Example 11;

[0058] Figure 7 is the processing graph of platelet aggregation sample 3 by using the self-made reagent and the commercially available preparation in the PLT-F channel in Example 11;

[0059] Figure 8 is the processing graph of platelet aggregation sample 4 by using the self-made reagent and the commercially available preparation in the PLT-F channel in Example 11. Detailed implementation mode

[0060] For a clearer understanding of the technical features, objectives and effects of the present invention, the detailed implementation mode of the present invention will be described in detail below.

[0061] An asymmetric cyanine compound has the following general structural formula I:

[0062]

[0063] Wherein:

[0064] Z is C(CH 3 ) 2 , O, S or Se.

[0065] R 1 and R 2 each independently selected from H or C 1-6 alkyl; R 1 and R 2 can be the same or different, R 1 and R 2 can each be selected from H, or can each be selected from alkyl groups having 1, 2, 3, 4, 5 or 6 carbons, and are not specifically limited.

[0066] R3 is H, C 1-10 saturated alkyl, C 3-18 alkenyl and C 3-18Alkynyl; R3 can be H, or can also be selected from saturated alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons, without specific limitation; it can also be selected from alkenyl groups having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons; it can also be selected from alkynyl groups having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons.

[0067] R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 , -C 1-18 alkyl-OR 6 or halogen; R 4 and R5 2 can each be selected from H, or can each be selected from alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons, without specific limitation. They can also each be selected from OR 6 , can also each be selected from halogen, can also be selected from -C 1 alkyl-OR 6 , -C 2 alkyl-OR 6 , -C 3 alkyl-OR 6 , -C 4 alkyl-OR 6 , -C 5 alkyl-OR 6 , -C 6 alkyl-OR 6 , -C 7 alkyl-OR 6 , -C 8 alkyl-OR 6 , -C 9 alkyl-OR 6 , -C 10 alkyl-OR 6 , -C 11 -OR 6 , -C 12 alkyl-OR 6 , -C 13 alkyl-OR 6 , -C 14 alkyl-OR 6 , -C 15 alkyl-OR 6 , -C 16 alkyl-OR 6 , -C 17 alkyl-OR 6, or -C 18 alkyl-OR 6 ; wherein, R 6 is C 2-18 alkyl, optionally selected from alkyl groups having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 carbons, and is not specifically limited.

[0068] X- is an anion, which can be any suitable negative ion, including but not limited to inorganic anions or organic anions, such as fluorine, chlorine, bromine and iodine, etc.

[0069] The term "alkyl" as used herein includes straight-chain alkyl and branched-chain alkyl. When referring to a single alkyl group such as "propyl", it specifically refers only to the straight-chain alkyl. When referring to a single branched-chain alkyl group such as "isopropyl", it specifically refers only to the branched-chain alkyl.

[0070] The term "halogen" as used herein includes but not limited to fluorine, chlorine, bromine and iodine.

[0071] In a specific embodiment, R 1 and R 2 each independently selected from H or C 2-4 alkyl; R 4 and R 5 each independently selected from H, C 1-4 alkyl, OR 6 , -C 5-9 alkyl-OR 6 or halogen; R 6 is C 3-10 alkyl.

[0073] In a specific embodiment, X - is selected from anions of halide ions, perchlorate, hexafluorophosphate or p-toluenesulfonate, including but not limited to inorganic anions or organic anions, and halide ions include but not limited to fluoride ions, chloride ions, bromide ions and iodide ions, etc.

[0074] In some specific embodiments, the asymmetric cyanine compounds are selected from

[0075]

[0076]

[0077]

[0078] The compounds of the present invention can be directly used for staining biological samples in the form of salts herein. Alternatively, the compounds of the present invention can be used in the form of derivatives of the compounds of Formula I, and the derivatives include but are not limited to conjugates. As used herein, "conjugate" refers to a compound formed by covalently linking a compound of the general formula of the present invention with other molecules.

[0079] The present invention also provides a composition comprising the above-mentioned compound of Formula I, the composition is composed of at least two compounds combined, and the composition is used for staining platelets.

[0080] The present invention also provides a method for preparing an asymmetric cyanine compound, comprising the following steps:

[0081] S1. Reacting a compound of General Formula II with an R 3 X compound to obtain a first quaternary ammonium salt intermediate III;

[0082]

[0083] In step S1, the reaction temperature is 50 - 160 °C, and the reaction time is 4 - 36 hours. For example, the reaction temperatures are 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C, 110 °C, 120 °C, 130 °C, 140 °C, 150 °C and 160 °C, etc., and the reaction times are 4 h, 5 h, 8 h, 10 h, 12 h, 15 h, 18 h, 20 h, 25 h, 28 h, 30 h, 32 h, 35 h and 36 h, etc., and specific values are not limited; the reaction solvent is selected from at least one polar solvent of dichloromethane, ethanol, acetonitrile, ethyl acetate, toluene, xylene, and the molar ratio of the compound of General Formula II to the R 3 X compound is 1:(1 - 10), and the molar ratio of the two is 1:1, 1:2, 1:4, 1:5, 1:8, 1:10, etc., and specific values are not limited.

[0084] S2. Reacting a 4-methylquinoline aromatic heterocyclic compound of Formula IV with 1,4-dibromobutane to obtain a second quaternary ammonium salt intermediate V.

[0085]

[0086] In step S2, the reaction temperature is 50 - 160°C, and the reaction time is 4 - 36 hours. For example, the reaction temperatures can be 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, and 160°C, etc.; the reaction times can be 4h, 5h, 8h, 10h, 12h, 15h, 18h, 20h, 25h, 28h, 30h, 32h, 35h, and 36h, etc., without specific limitations. The reaction solvent is selected from at least one polar solvent among dichloromethane, ethanol, acetonitrile, ethyl acetate, toluene, and xylene. The molar ratio of the compound of formula IV to 1,4 - dibromobutane is 1:(1 - 5); the molar ratios can be 1:1, 1:2, 1:4, 1:5, etc., without specific limitations.

[0087] S3. Condense the first - stage quaternary ammonium salt intermediate III obtained in step S1 with N,N’ - diphenylformamidine to obtain the compound of formula VI.

[0088]

[0089] In step S3, the reaction temperature is 100 - 250°C, and the reaction time is 20 minutes to 2 hours. For example, the reaction temperatures can be 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, etc.; the reaction times can be 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min, etc., without specific limitations. The reaction solvent is acetic anhydride, acetic acid, or a mixture thereof. The molar ratio of the first - stage quaternary ammonium salt intermediate III to N,N’ - diphenylformamidine is 1:(1.2 - 2), and the molar ratios can be 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, etc., without specific limitations.

[0090] S4. React the second - stage quaternary ammonium salt intermediate of formula V obtained in step S2 with the compound of formula VI obtained in step S3 to obtain the compound of formula VII.

[0091]

[0092] In step S4, the reaction temperature is 10 - 160 °C, and the reaction time is 30 minutes - 5 hours. For example, the reaction temperature can be 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C, 110 °C, 120 °C, 130 °C, 140 °C, 150 °C, and 160 °C, etc., and the reaction time can be 20 min, 30 min, 40 min, 50 min, 60 min, 2 h, 3 h, 4 h, 5 h, etc., without specific limitation; the reaction solvent is selected from at least one polar organic solvent such as dichloromethane, chloroform, acetonitrile, ethyl acetate, methanol, and ethanol, the catalyst is a mixture of acetic anhydride and an organic base, and the molar ratio of the quaternary ammonium salt intermediate of formula V to the compound of formula VI is 1:2 - 2:1. The molar ratios such as 1:2, 1:1.4, 1:1, 2:1, etc. are not specifically limited.

[0093] S5. React the compound VII obtained in step S4 with HN(R 1 R 2 ) to obtain the asymmetric cyanine compound of formula I;

[0094]

[0095] In step S5, the reaction temperature is 50 - 180 °C, and the reaction time is 30 minutes - 10 hours. For example, the reaction temperature can be 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C, 110 °C, 120 °C, 130 °C, 140 °C, 150 °C, and 160 °C, etc., and the reaction time can be 30 min, 40 min, 50 min, 60 min, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, and 10 h, etc., without specific limitation; the reaction solvent is selected from at least one organic solvent such as dichloromethane, acetonitrile, methanol, ethanol, and ethylene glycol monomethyl ether; the molar ratio of compound VII to HN(R 1 R 2 ) is 1:2 - 1:30. The molar ratios such as 1:2, 1:4, 1:10, 1:30, etc. are not specifically limited.

[0096] Wherein: Z is C(CH 3 ) 2 , O, S or Se;

[0097] R 1 and R 2 each independently selected from H or C 1-6 alkyl;

[0098] R3 is H, C 1-10 saturated alkyl, C 3-18 alkenyl or C 3-18 alkynyl;

[0099] R4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 , -C 1-18 alkyl-OR 6 or halogen;

[0100] R 6 is C 2-18 alkyl;

[0101] X - is an anion.

[0102] The present invention also provides a kit for platelet determination, which includes a diluent and a fluorescent staining solution. The pH of the diluent is 8-10. The diluent includes a buffer system, inorganic salts, a surfactant and a preservative. The fluorescent staining solution includes an asymmetric cyanine compound. The fluorescent staining solution may also include solvents such as isopropanol and ethylene glycol.

[0103] The concentration of the asymmetric cyanine compound of the present invention in the fluorescent staining solution is 0.005-1 g / L. For example, the concentration of the asymmetric cyanine compound is controlled to be 0.005 g / L, 0.01 g / L, 0.015 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L and 1 g / L, etc., and no specific limitation is made. The platelet nucleic acid dye formed by the asymmetric cyanine compound of the present invention is specific to platelet nucleic acid; the wavelength difference between the maximum emission wavelength of the free staining solution formed by the asymmetric cyanine compound of the present invention and the maximum emission wavelength after the dye binds to nucleic acid is greater than 10 nm. The luminescence of the free staining solution can be filtered by a filter, reducing the overlap of the fluorescence spectra and reducing the interference of the fluorescence of the free state staining solution on the sample detection (fluorescence of the bound state dye). When a filter is added to filter the emitted light and the fluorescence of the free state dye, the filter will not filter the fluorescence of the dye after binding to nucleic acid, retaining the fluorescence intensity of the staining solution bound to nucleic acid in cells to the greatest extent and improving the light utilization rate. At the same time, after the dye binds to nucleic acid, its fluorescence intensity is greatly enhanced compared with that of the free state fluorescent staining solution, improving the signal-to-noise ratio of the detection.

[0104] The nucleic acid fluorescent staining solution formed by the symmetric cyanine compound of the present invention and the semiconductor laser flow cytometry can accurately count platelets in the PLT-F channel. By treating the platelet morphology with the diluent, while shaping it, the cell membrane morphology of each type is changed, facilitating the specific binding of platelet nucleic acid to the fluorescent staining solution respectively, thereby realizing the counting of platelets. In the present invention, the nucleic acid fluorescent staining solution with an amino group and the diluent act together to achieve the depolymerization of aggregated platelets, avoiding the interference of aggregated platelets on the detection in the PLT-F channel and accurately counting platelet aggregation samples.

[0105] To ensure that the pH value range of the dilution is stably maintained between 8 and 10, a buffer system can be used for adjustment. Therefore, the dilution includes a buffer system for maintaining the pH value, and the buffer system is selected from at least one of a phosphate buffer system, an ammonium chloride buffer system, and a glycine buffer system. Preferably, the concentration of the buffer system in the dilution is 0.1 g - 10 g / L. For example, the concentration can be controlled at 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, etc., and no specific limitation is made.

[0106] Furthermore, it is preferred that the surfactant is at least one of a non-ionic surfactant, an amphoteric surfactant, a cationic surfactant, and an anionic surfactant, and the concentration of the surfactant in the diluent is 0.005-10 g / L; for example, the concentration of the surfactant is controlled to be 0.005 g / L, 0.01 g / L, 0.015 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, etc., and no specific limitation is made. Commonly used cationic surfactants are quaternary ammonium salt cationic surfactants. Quaternary ammonium salt cationic surfactants include at least one of dodecyltrimethylammonium bromide (DTAB), decyltrimethylammonium bromide (CTAB), and octyltrimethylammonium bromide (OTAB). The use concentration of the cationic surfactant is 0.1 g-10 g / L. For example, the concentration of the cationic surfactant is controlled to be 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, etc., and no specific limitation is made. Among them, it is preferred that the concentration of OTAB is 1 g-10 g / L, such as 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, etc., and no specific limitation is made; the concentration of CTAB is 0.5 g-5 g / L, such as 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, etc., and no specific limitation is made; the concentration of DTAB is 0.1 g-3 g / L, such as 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, etc., and no specific limitation is made; as the total number of carbon atoms of the cationic surfactant increases, the usage amount decreases. Amphoteric surfactants can be selected from betaine-type amphoteric surfactants, and commonly used ones are at least one of lauryl betaine, tetradecyl betaine, and lauramidopropyl betaine. The use concentration of the amphoteric surfactant is 0.1 g-5 g / L, such as 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, etc., and no specific limitation is made.The non-ionic surfactant can be a polyoxyethylene-type non-ionic surfactant. The concentration of the non-ionic surfactant is 0.1 - 5 g / L, such as 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, etc., without specific limitation. Preferably, it is 0.1 - 2 g / L. Commonly used polyoxyethylene-type non-ionic surfactants include cetyl polyethylene glycol ether (n = 20), cetyl polyethylene glycol ether (n = 30), polyethylene glycol monostearate (n = 25), and polyoxyethylene oleyl ether (n = 20). The non-ionic surfactant can play a role in dispersion and solubilization.

[0107] Further, it is preferred that the concentration of the preservative in the diluent is 0.5 - 4 g / L, such as 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, etc., without specific limitation. The preservative includes at least one of benzoic acid and its salts, isothiazolinone, sodium hydroxymethylglycinate, phenoxyethanol, and benzyl alcohol. The preservative can effectively control the growth of microorganisms in the reagent and extend the validity period of the reagent kit.

[0108] Further, it is preferred that the inorganic salt is one or more of sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, disodium ethylenediaminetetraacetate, sodium carbonate, and sodium bicarbonate. The concentration of the inorganic salt in the diluent is 0.1 g - 10 g / L, such as controlling the concentration to be 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, etc., without specific limitation. The addition of the inorganic salt can make the osmotic pressure of the detection reagent of the present invention within the range suitable for human physiological osmotic pressure, and adjust the osmotic pressure concentration to physiological osmotic pressure (for example, 150 - 600 mOsm / kg).

[0109] Example 1

[0110] An asymmetric cyanine compound 1 has the following chemical structural formula:

[0111]

[0112] The preparation method of the asymmetric cyanine compound 1 is as follows: 10 mmol of 1-ethyl-4-methylthiazolium quaternary salt and 20 mmol of N,N-dimethylformamidine are heated and stirred in 30 mL of acetic acid solvent on an oil bath at 100 °C for 1.5 hours. After cooling, the orange-yellow solid precipitated from the reaction solution is filtered, washed with n-hexane, and 5.4 g of the orange solid is recrystallized in 50 mL of ethanol. The orange solid is filtered and dried, and the yield is 41.5%. 10 mmol of methylquinoline quaternary salt, 2 mL of triethylamine and 2 mL of acetic anhydride are added to the orange solid, and the reaction is carried out at room temperature in the dark for 1.5 hours in 20 mL of dichloromethane / methanol (v / v = 1:1). The reaction solution is poured into ether, and blue small particle products are precipitated. 5 mmol of the blue compound and 50 mmol of the organic amine HN(R 1 R 2 ) are dissolved in 10 mL of ethylene glycol monomethyl ether and heated for reaction for 1.5 hours. The reaction solution is poured into 100 mL of ether to precipitate a blue solid, which is separated by a basic silica gel column and eluted with a gradient of dichloromethane and methanol as the eluent. The blue fraction is collected to obtain compound 1 with a yield of 54%.

[0113] Example 2

[0114] An asymmetric cyanine compound 2 has the following chemical structural formula:

[0115]

[0116] The preparation method of the asymmetric cyanine compound 2 is as follows: 10 mmol of 1-propenyl-4-methylthiazolium quaternary salt and 20 mmol of N,N-dimethylformamidine are heated and stirred in 30 mL of acetic acid solvent on an oil bath at 100 °C for 1.5 hours. After cooling, the orange-yellow solid precipitated from the reaction solution is filtered, washed with n-hexane, and 5.0 g of the orange-red solid is recrystallized in 50 mL of ethanol. The orange solid is filtered and dried, and the yield is 39%. 10 mmol of methylquinoline quaternary salt, 2 mL of triethylamine and 2 mL of acetic anhydride are added to the orange solid, and the reaction is carried out at room temperature in the dark for 1.5 hours in 20 mL of dichloromethane / methanol (v / v = 1:1). The reaction solution is poured into ether, and blue small particle products are precipitated. 5 mmol of the blue compound and 50 mmol of the organic amine HN(R 1 R 2 ) are dissolved in 10 mL of ethylene glycol monomethyl ether and heated for reaction for 1.0 hour. The reaction solution is poured into 100 mL of ether to precipitate a blue solid, which is separated by a basic silica gel column and eluted with a gradient of dichloromethane and methanol as the eluent. The blue fraction is collected to obtain compound 2 with a yield of 61%.

[0117] Example 3

[0118] An asymmetric cyanine compound 3 has the following chemical structural formula:

[0119]

[0120] The preparation method of the asymmetric cyanine compound 3 is as follows: 10 mmol of 1-butynyl-4-methylthiazolium quaternary ammonium salt and 20 mmol of N,N-dimethylformamidine are heated and stirred in 30 mL of acetic acid solvent on an oil bath at 100 °C for 2 hours. After cooling, the orange-yellow solid precipitated from the reaction solution is filtered, washed with n-hexane, and 4.6 g of the orange solid is recrystallized in 50 mL of ethanol, and the orange solid is filtered and dried with a yield of 35%. 10 mmol of methylquinoline quaternary ammonium salt, 2 mL each of triethylamine and acetic anhydride are added to the orange solid, and the reaction is carried out at room temperature in the dark for 1.5 hours in 20 mL of dichloromethane / methanol (v / v = 1:1). The reaction solution is poured into ether, and blue small particle products are precipitated. 5 mmol of the blue compound and 50 mmol of the organic amine HN(R 1 R 2 ) are dissolved in 10 mL of ethylene glycol monomethyl ether and heated for reaction for 2 hours. The reaction solution is poured into 100 mL of ether to precipitate a blue solid, which is separated by an alkaline silica gel column and eluted with a gradient of dichloromethane and methanol as the eluent, and the blue fraction is collected to obtain compound 3 with a yield of 46%.

[0121] Example 4

[0122] An asymmetric cyanine compound 4 has the following chemical structural formula:

[0123]

[0124] The preparation method of the asymmetric cyanine compound 4 is as follows: 10 mmol of 1-butyl-4-methylthiazolium quaternary ammonium salt and 20 mmol of N,N-dimethylformamidine are heated and stirred in 30 mL of acetic acid solvent on an oil bath at 100 °C for 1.5 hours. After cooling, the orange-yellow solid precipitated from the reaction solution is filtered, washed with n-hexane, and 6 g of the orange solid is recrystallized in 50 mL of ethanol, and the orange solid is filtered and dried with a yield of 60%. 10 mmol of methylquinoline quaternary ammonium salt, 2 mL each of triethylamine and acetic anhydride are added to the orange solid, and the reaction is carried out at room temperature in the dark for 1.5 hours in 20 mL of dichloromethane / methanol (v / v = 1:1). The reaction solution is poured into ether, and blue small particle products are precipitated. 5 mmol of the blue compound and 50 mmol of the organic amine HN(R 1 R 2 ) are dissolved in 10 mL of ethylene glycol monomethyl ether and heated for reaction for 0.5 hours. The reaction solution is poured into 100 mL of ether to precipitate a blue solid, which is separated by an alkaline silica gel column and eluted with a gradient of dichloromethane and methanol as the eluent, and the blue fraction is collected to obtain compound 4 with a yield of 65%.

[0125] Example 5

[0126] An asymmetric cyanine compound 5, the chemical structural formula is as follows:

[0127]

[0128] The preparation method of the asymmetric cyanine compound 5 is as follows: 10 mmol of 1-butenyl-4-methylthiazolium quaternary ammonium salt and 25 mmol of N,N-dimethylformamidine are heated and stirred in 30 mL of acetic acid solvent on an oil bath at 100 °C for 2.5 hours. After cooling, the orange-yellow solid precipitated from the reaction solution is filtered, washed with n-hexane, and 5.6 g of the orange solid is recrystallized in 50 mL of ethanol, and the orange solid is filtered and dried, with a yield of 43%. 10 mmol of methylquinoline quaternary ammonium salt, 2 mL each of triethylamine and acetic anhydride are added to the orange solid, and the reaction is carried out in the dark at room temperature for 1.5 hours in 20 mL of dichloromethane / methanol (v / v = 1:1). The reaction solution is poured into ether, and blue small particle products are precipitated. 5 mmol of the blue compound and 50 mmol of the organic amine HN(R 1 R 2 ) are dissolved in 10 mL of ethylene glycol monomethyl ether and heated to react for 1.5 hours. The reaction solution is poured into 100 mL of ether to precipitate a blue solid, which is separated by an alkaline silica gel column and eluted with a gradient of dichloromethane and methanol as the eluent, and the blue fraction is collected to obtain compound 5 with a yield of 51%.

[0129] Example 6

[0130] An asymmetric cyanine compound 6, the chemical structural formula is as follows:

[0131]

[0132] The preparation method of the asymmetric cyanine compound 6 is as follows: 10 mmol of 1-pentynyl-4-methylthiazolium quaternary ammonium salt and 20 mmol of N,N-dimethylformamidine are heated and stirred in 30 mL of acetic acid solvent on an oil bath at 100 °C for 1.0 hour. After cooling, the orange-yellow solid precipitated from the reaction solution is filtered, washed with n-hexane, and 4.9 g of the orange solid is recrystallized in 50 mL of ethanol, and the orange solid is filtered and dried, with a yield of 38%. 10 mmol of methylquinoline quaternary ammonium salt, 2 mL each of triethylamine and acetic anhydride are added to the orange solid, and the reaction is carried out in the dark at room temperature for 1.5 hours in 20 mL of dichloromethane / methanol (v / v = 1:1). The reaction solution is poured into ether, and blue small particle products are precipitated. 5 mmol of the blue compound and 50 mmol of the organic amine HN(R 1 R 2 ) are dissolved in 10 mL of ethylene glycol monomethyl ether and heated to react for 1.5 hours. The reaction solution is poured into 100 mL of ether to precipitate a blue solid, which is separated by an alkaline silica gel column and eluted with a gradient of dichloromethane and methanol as the eluent, and the blue fraction is collected to obtain compound 6 with a yield of 39%.

[0133] Example 7

[0134] An asymmetric cyanine compound 7 has the following chemical structural formula:

[0135]

[0136] The preparation method of the asymmetric cyanine compound 7 is as follows: 10 mmol of 1-ethyl-4-methylthiazolium quaternary salt and 15 mmol of N,N–dimethylformamidine are heated and stirred in 30 mL of acetic acid solvent on an oil bath at 100 °C for 1.0 hour. After cooling, the orange-yellow solid precipitated from the reaction solution is filtered, washed with n-hexane, and 5.5 g of the orange solid is recrystallized in 50 mL of ethanol, filtered and dried. The yield is 43%. 10 mmol of methylquinoline quaternary salt, 2 mL each of triethylamine and acetic anhydride are added to the orange solid, and the reaction is carried out at room temperature in the dark for 1.5 hours in 20 mL of dichloromethane / methanol (v / v = 1:1). The reaction solution is poured into ether, and blue small particle products are precipitated. 5 mmol of the blue compound and 50 mmol of organic amine HN(R 1 R 2 ) are dissolved in 10 mL of ethylene glycol monomethyl ether and heated for reaction for 1.0 hour. The reaction solution is poured into 100 mL of ether to precipitate a blue solid, which is separated by a basic silica gel column and eluted with a gradient of dichloromethane and methanol as the eluent. The blue fraction is collected to obtain compound 7 with a yield of 46%.

[0137] Example 8

[0138] An asymmetric cyanine compound 8 has the following chemical structural formula:

[0139]

[0140] The preparation method of the asymmetric cyanine compound 8 is as follows: 10 mmol of 1-butenyl-4-methylthiazolium quaternary salt and 20 mmol of N,N–dimethylformamidine are heated and stirred in 30 mL of acetic acid solvent on an oil bath at 100 °C for 2 hours. After cooling, the orange-yellow solid precipitated from the reaction solution is filtered, washed with n-hexane, and 5.2 g of the orange solid is recrystallized in 50 mL of ethanol, filtered and dried. The yield is 40%. 10 mmol of methylquinoline quaternary salt, 2 mL each of triethylamine and acetic anhydride are added to the orange solid, and the reaction is carried out at room temperature in the dark for 1.5 hours in 20 mL of dichloromethane / methanol (v / v = 1:1). The reaction solution is poured into ether, and blue small particle products are precipitated. 5 mmol of the blue compound and 50 mmol of organic amine HN(R 1 R 2) It was dissolved in 10 mL of ethylene glycol monomethyl ether and heated for reaction for 1.0 h. The reaction solution was poured into 100 mL of diethyl ether to precipitate a blue solid, which was separated by an alkaline silica gel column and eluted with a gradient of eluents dichloromethane and methanol. The blue fraction was collected to obtain Compound 8 with a yield of 67%.

[0141] Example 9

[0142] An asymmetric cyanine compound 9 has the following chemical structural formula:

[0143]

[0144] The preparation method of the asymmetric cyanine compound 9 is as follows: 10 mmol of 1-butynyl-4-methylthiazolium quaternary salt and 20 mmol of N,N-dimethylformamidine were heated and stirred in 30 mL of acetic acid solvent on an oil bath at 100 °C for 1.0 h. After cooling, the orange-yellow solid precipitated from the reaction solution was filtered, washed with n-hexane, and 6.0 g of the orange solid was recrystallized from 50 mL of ethanol. The orange solid was filtered and dried with a yield of 52%. 10 mmol of methylquinoline quaternary salt, 2 mL of triethylamine and 2 mL of acetic anhydride were added to the orange solid, and the reaction was carried out at room temperature in the dark for 1.5 h in 20 mL of dichloromethane / methanol (v / v = 1:1). The reaction solution was poured into diethyl ether to precipitate blue small particle products. 5 mmol of the blue compound and 50 mmol of organic amine HN(R 1 R 2 ) were dissolved in 10 mL of ethylene glycol monomethyl ether and heated for reaction for 1.0 h. The reaction solution was poured into 100 mL of diethyl ether to precipitate a blue solid, which was separated by an alkaline silica gel column and eluted with a gradient of eluents dichloromethane and methanol. The blue fraction was collected, and 100 mmol of sodium perchlorate salt was added for anion replacement to obtain Compound 9 with a yield of 67%.

[0145] Example 10

[0146] The fluorescence emission spectra and relative fluorescence intensities before and after the binding of commercially available dye Nile blue dye solution and self-made fluorescent dye A (asymmetric cyanine compound 1 in Example 1) to calf thymus DNA were measured. The specific operations are as follows:

[0147] Nile blue (Nile blue sulfate, CAS: 3625-57-8) can be purchased from the market, for example, from companies such as Aladdin Reagent Company and Merck.

[0148] An EG (ethylene glycol) solution of Compound 1 with a concentration of 0.008 g / L (also called Dye A) was prepared. 25 μL was taken and diluted to 1 mL with a pH 7.4, 10 mM phosphate buffer solution (PBS solution), placed in a cuvette, and its fluorescence intensity was measured.

[0149] Prepare a PBS solution containing a certain concentration of calf thymus DNA. Take another 25 μL of the EG (ethylene glycol) solution of dye A with a concentration of 0.008 g / L into a cuvette, then add the PBS solution of calf thymus DNA to the solution, and finally dilute it to 1 mL with a tris(hydroxymethyl)aminomethane hydrochloride buffer solution with pH 7.4 and 10 mM. Measure the fluorescence intensities of the PBS solutions of calf thymus DNA with final concentrations of 5 μg / mL and 10 μg / mL respectively. Fluorescence spectrophotometer, model: HITACHI F-7100.

[0150] The specific detection results are shown in the appendix Figure 1 and Figure 2 , from Figure 1 it can be seen that after dye A binds to 10 μg / mL calf thymus DNA, the relative fluorescence intensity of the fluorescence spectrum emission peak at 663 nm increases by 23.79 times (I / I 0 = 53 / 3.66 = 1.17). From Figure 2 it can be seen that after Nile blue dye binds to 10 μg / mL calf thymus DNA, the relative fluorescence intensity of the fluorescence spectrum emission peak at 677 nm increases by 23.79 times (I / I 0 = 21.06 / 17.96 = 1.17).

[0151] From Figure 3 it can be seen that the maximum emission wavelength of free dye A (unbound to calf thymus DNA) is 663.4 nm. When dye A binds to 10 μg / mL calf thymus DNA, the maximum emission wavelength shifts 12 nm to the right to 651.4 nm. In a hematology analyzer, when using a filter to filter the light emission of free dye, the overlap of the fluorescence spectrum can be reduced, and the bound fluorescence intensity can be better retained, thereby improving the light utilization rate. From Figure 4 it can be seen that the maximum emission wavelengths of the free state and bound state of Nile blue dye are the same and there is no wavelength difference. This makes the free dye easily interfere with the detection.

[0152] Example 11

[0153] A kit for platelet determination, comprising diluent A and a fluorescent dye. The specific components are shown in Table 1. The asymmetric cyanine dye in Table 1 is selected from Compound 1 of Example 1, Compound 2 of Example 2, and Compound 3 of Example 3, and dye A, dye B, and dye C are obtained respectively.

[0154] Table 1. Composition of the kit of Example 11

[0155]

[0156]

[0157] After preparing the dilution solution and the fluorescent staining solution according to the above table and making up to the specified volume, stir evenly at room temperature and filter using a 0.22 μm filter membrane. Compare the detection results of aggregated platelets between the reagent (nile blue staining solution + dilution solution) in the PLT-F channel of the Sysmex XN1000 instrument and the self-made reagent (dilution solution + staining solution) in the PLT-F channel of Example 11. The detailed results are shown in Table 2 and Table 3.

[0158] Table 2. Comparison of test results of nile blue staining solution and self-made reagent in the PLT-F channel of Example 11 for normal samples

[0159]

[0160] Table 3. Comparison of test results of nile blue staining solution and self-made reagent in the PLT-F channel of Example 11 for normal samples

[0161]

[0162] The self-made reagent (dilution solution A and staining solution A) in Example 11 and the commercially available preparation (dilution solution A + nile blue staining solution) were used to process the above platelet aggregation samples 1 - 4 respectively. The platelet processing graphs in the PLT-F channel are shown in Figures 5 - 8 , where the left side is the result graph of the commercially available preparation (dilution solution A + nile blue staining solution) processing the same sample, and the right side is the result graph of the self-made reagent (dilution solution A and staining solution A) processing the same sample; as can be seen from the figure, the commercially available preparation (dilution solution A + nile blue staining solution) cannot depolymerize platelets. The nucleic acid fluorescent staining solution and the dilution solution in the present invention act together to achieve the depolymerization of aggregated platelets, avoid the interference of aggregated platelets on the detection of the PLT-F channel, and can accurately count platelet aggregation samples.

[0163] From the detection results in Table 2 and Table 3, by comparing the counting accuracy of the asymmetric cyanine fluorescent staining solution A and the supporting dilution solution A nile blue dye for normal samples, the supporting reagent of the present invention accurately counts the number of platelets in normal samples. At the same time, the counting results of the platelet fluorescence channel (PLT-F) using the supporting reagent of the present invention are compared with those of the impedance channel (PLT-I), and the results are consistent. For aggregated samples, since the PLT-I channel does not have the depolymerization function, its counting result is the platelet result after sample aggregation. Comparing the PLT-I and PLT-F results of the nile blue staining solution, the nile blue staining solution does not have the depolymerization function, while the supporting reagent of the present invention can achieve the depolymerization of aggregated samples.

[0164] Example 12

[0165] A kit for platelet determination, comprising diluent B and fluorescent dye solution. The specific components are shown in Table 4. The asymmetric cyanine dyes in Table 4 are selected from Compound 1 of Example 1, Compound 2 of Example 2, and Compound 3 of Example 3, and Dye Solution A, Dye Solution B, and Dye Solution C are obtained respectively.

[0166] Table 4. Composition of the kit of Example 12

[0167]

[0168] After preparing the diluent and the dye solution according to the above table and making up the volume to the specified amount, stir evenly at room temperature and filter using a 0.22 μm filter membrane. Compare the Nile blue dye solution of the PLT-F channel of the Sysmex XN1000 instrument with the self-made reagent of the PLT-F channel of Example 12 for the detection results of normal samples and aggregated platelets. The detailed results are shown in Table 5 and Table 6.

[0169] Table 5. Comparison of test results of normal samples between Nile blue dye solution and self-made reagent of the PLT-F channel of Example 12

[0170]

[0171]

[0172] Table 6. Comparison of detection results of platelet aggregation between Nile blue dye solution and self-made reagent of the PLT-F channel of Example 12

[0173]

[0174] From the results in Table 5 and Table 6, by comparing the counting accuracy of the normal samples by the asymmetric cyanine fluorescent Dye Solution A and the supporting diluent A Nile blue dye, the supporting reagent of the present invention accurately counts the number of platelets in normal samples. At the same time, the counting results of the platelet fluorescence channel (PLT-F) using the supporting reagent of the present invention are compared with those of the impedance channel (PLT-I), and the results are consistent. For aggregated samples, since the PLT-I channel does not have a depolymerization function, its counting result is the platelet result after sample aggregation. By comparing the PLT-I and PLT-F results of the Nile blue dye solution, the Nile blue dye solution does not have a depolymerization function, while the supporting reagent of the present invention can achieve the depolymerization of aggregated samples.

[0175] The above data and attached drawings indicate that when the compounds of the present invention, the preparation methods of the compounds, the fluorescent staining solutions, and the detection reagents are used for relevant performance tests, the obtained results all meet the acceptance criteria. The feasibility and rationality of the present invention are verified through the above specific embodiments. These embodiments are only illustrative of individual alternative embodiments of the present invention and are not limited thereto. For those skilled in the art, any changes, modifications, and substitutions made according to the scope of the patent application of the present invention should be included within the scope of the claims of this patent and all fall within the protection scope of the present invention.

Claims

1. An asymmetric cyanine compound, characterized in that, it has the following general structural formula I: wherein: Z is C(CH 3 ) 2 , O, S or Se; R 1 and R 2 each independently selected from H or C 1-6 alkyl; R3 is H, C 1-10 a saturated alkyl group, C 3-18 an alkenyl group or C 3-18 an alkynyl group; R 4 and R 5 each independently selected from H, C 1-18 alkyl, OR 6 , -C 1-18 alkyl-OR 6 or halogen; R 6 is C 2-18 alkyl; X - is an anion.

2. The asymmetric cyanine compound according to claim 1, characterized in that, R 1 and R 2 each independently selected from C 2-4 alkyl; R 4 and R 5 are each independently selected from H, C 1-4 alkyl, OR 6 , -C 5-9 alkyl-OR 6 or halogen; R 6 is C 3-10 alkyl 3. The asymmetric cyanine compound according to claim 1, characterized in that, X - An anion selected from a halide ion, perchlorate, hexafluorophosphate or p-toluenesulfonate.

4. The asymmetric cyanine compound according to claim 1, characterized in that, the asymmetric cyanine compound is at least one of the following compounds:

5. A preparation method of the asymmetric cyanine compound according to any one of claims 1-4, characterized in that, it includes the following steps: S1. The first quaternary ammonium salt intermediate III is prepared by reacting a compound of general formula II with an R 3 compound X; S2. Reacting the 4-methylquinoline aromatic heterocyclic compound of formula IV with 1,4-dibromobutane to obtain the second quaternary ammonium salt intermediate V; S3. Condensing the first quaternary ammonium salt intermediate III obtained in step S1 with N,N'-diphenylformamidine to obtain the compound of formula VI; S4. Reacting the second quaternary ammonium salt intermediate V of formula V obtained in step S2 and the compound of formula VI obtained in step S3 to obtain the compound of formula VII; S5. React the VII compound obtained in step S4 with HN(R 1 R 2 ) to obtain the asymmetric cyanine compound of formula I; wherein: Z is C(CH 3 ) 2 , O, S or Se; R 1 and R 2 each independently selected from H or C 1-6 alkyl; R3 is H, C 1-10 a saturated alkyl group, C 3-18 an alkenyl group or C 3-18 an alkynyl group; R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 , -C 1-18 alkyl-OR 6 or halogen; R 6 is C 2-18 alkyl; X - is an anion.

6. The preparation method of the asymmetric cyanine compound according to claim 5, characterized in that, In step S1, the reaction temperature is 50 - 160 °C, the reaction time is 4 - 36 hours, the reaction solvent is selected from at least one polar solvent among dichloromethane, ethanol, acetonitrile, ethyl acetate, toluene, and xylene, and the molar ratio of the compound of general formula II to the compound R 3 X is 1:(1 - 10); in step S2, the reaction temperature is 50-160 °C, the reaction time is 4-36 hours, the reaction solvent is selected from at least one polar solvent of dichloromethane, ethanol, acetonitrile, ethyl acetate, toluene, xylene, and the feeding molar ratio of the compound of formula IV to 1,4-dibromobutane is 1:(1-5); in step S3, the reaction temperature is 100-250 °C, the reaction time is 20 minutes to 2 hours, the reaction solvent is acetic anhydride, acetic acid or a mixture thereof, and the feeding molar ratio of the first quaternary ammonium salt intermediate III to N,N'-diphenylformamidine is 1:(1.2-2); in step S4, the reaction temperature is 10-160 °C, the reaction time is 30 minutes - 5 hours, the reaction solvent is selected from at least one polar organic solvent of dichloromethane, chloroform, acetonitrile, ethyl acetate, methanol, ethanol, the catalyst is a mixture of acetic anhydride and an organic base, and the feeding molar ratio of the second quaternary ammonium salt intermediate V of formula V to the compound of formula VI is 1:2 - 2:1; In step S5, the reaction temperature is 50 - 180 °C, the reaction time is 30 minutes - 10 hours, and the reaction solvent is selected from at least one organic solvent among dichloromethane, acetonitrile, methanol, ethanol, and ethylene glycol monomethyl ether; the molar ratio of compound VII to HN(R 1 R 2 ) in the feed is 1:2 - 1:

30.

7. A kit for platelet determination, characterized in that, it includes a diluent and a fluorescent staining solution, the pH of the diluent is 8-10, the diluent includes a buffer system, inorganic salts, a surfactant and a preservative, and the fluorescent staining solution includes the asymmetric cyanine compound according to any one of claims 1-4.

8. The kit for platelet determination according to claim 7, characterized in that, the concentration of the asymmetric cyanine compound in the fluorescent staining solution is 0.005-1 g / L.

9. The kit for platelet determination according to claim 7, characterized in that, the concentration of the buffer system in the diluent is 0.1 g-10 g / L, and the buffer system is at least one of a phosphate buffer system, an ammonium chloride buffer system, and an aminoacetic acid buffer system.

10. The kit for platelet determination according to claim 7, characterized in that, The inorganic salt is one or more of sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, disodium ethylenediaminetetraacetate, sodium carbonate, and sodium bicarbonate, and the concentration of the inorganic salt in the diluent is 0.1 g - 10 g / L; The surfactant is at least one of nonionic surfactants, amphoteric surfactants, cationic surfactants, and anionic surfactants, and the concentration of the surfactant in the diluent is 0.005 - 10 g / L; The concentration of the preservative in the diluent is 0.5 - 5 g / L, and the preservative includes at least one of benzoic acid and its salts, isothiazolinone, sodium hydroxymethylglycinate, phenoxyethanol, and benzyl alcohol.

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