A fluorescent compound, a detection reagent, a kit for blood cell analysis

Abstract

The present application relates to the field of labeling reagents, in particular to a fluorescent compound, having any one of the following structural formula I to structural formula IV: (I), (II), (III), (IV). The present application also provides a detection reagent which can be used for staining a biological sample, comprising a dye reagent, wherein the dye reagent comprises the fluorescent compound. The present application also provides a kit comprising the detection reagent disclosed in the present application, which can be used for processing a blood sample, in particular a blood sample containing reticulocytes, to identify and count the reticulocytes.

Description

[0001] And its application in blood cell testing Technical Field This invention relates to the field of labeling reagents, and more specifically to a fluorescent compound, a detection reagent, a kit for blood cell analysis, and the application of this fluorescent compound in blood cell detection. Background Technology

[0002] Reticulocytes are the immature stage in the process of red blood cell maturation. Their number and maturation groups are core indicators for evaluating bone marrow hematopoietic function and are widely used in clinical fields such as differential diagnosis of various types of anemia, monitoring of bone marrow recovery after radiotherapy and chemotherapy, and evaluation of the efficacy of erythropoietin (EPO).

[0003] Currently, flow cytometry-based reticulocyte analysis technology is widely used in fully automated blood cell analysis systems. Its detection principle lies in the specific binding of fluorescent dyes to nucleic acid substances (mainly RNA) within reticulocytes. Upon laser excitation, a fluorescent signal is generated, which is detected by an optical system to identify and count reticulocytes. Because reticulocytes contain a significant amount of residual nucleic acid, their fluorescence intensity is significantly higher than that of mature red blood cells, allowing for effective differentiation.

[0004] However, despite the relatively mature technology, existing fluorescent dyes and staining methods still have the following obvious limitations: (1) Poor repeatability of staining effect: The staining results of most reported fluorescent dye molecules are easily affected by sample processing time, temperature and other conditions. The instrument control precision requirements are extremely high during testing, which easily leads to poor repeatability of sample test results; (2) High requirements for sample stability: Due to the different molecular structures of dyes, the binding regions with RNA are different. During the storage of blood samples, RNA will gradually degrade. If the dye binds to the degradation region of RNA molecules, it will be affected by the degradation region. As the storage time of the sample increases, the accuracy of the results may decrease significantly; (3) Limited resolution: Under the current dye conditions, it is difficult to achieve fine differentiation of different maturity stages of reticulocytes. In particular, the effective differentiation between higher maturity reticulocytes and normal red blood cells is insufficient, which restricts its application value in clinical classification.

[0005] Therefore, there is an urgent need to develop a new fluorescent dye and corresponding detection kit that can improve the tolerance to sample conditions and enhance the resolution while ensuring rapid and stable staining, thereby meeting the needs of modern clinical laboratories for automated, standardized and high-precision reticulocyte analysis. Summary of the Invention

[0006] The combination of small-molecule fluorescent probes and fluorescence imaging technology offers significant advantages such as high selectivity, high sensitivity, and high spatiotemporal resolution, and has been widely used in recent years for real-time detection of various bioactive molecules in cells and living organisms. However, traditional fluorescent probes (dyes) such as basic pigments like neomethylene blue (NMB) and brilliant tar blue (BCB) typically exhibit low quantum yields. Therefore, designing, developing, and synthesizing novel fluorescent compounds with high quantum yields is particularly important for the detection and quantification of blood cells.

[0007] Therefore, this invention designs and synthesizes a novel fluorescent compound having dimethylbenzimidazole, benzimidazole or silanized benzimidazole and pyridine, wherein the fluorescent compound has the following structural formula I: It has any one of the following structural formulas I to IV: In structural formulas I to IV, X is selected from any one of Si(CH3)2, NH, and N(CH3); m is an integer from 1 to 6; R1 is selected from substituted or unsubstituted phenyl, biphenyl, thiazolyl, thiophene, imidazolyl, thiazinyl, -NHR5, -NR6R7, wherein R5, R7 and R8 are each independently selected from H or C1-C8 straight-chain or branched alkyl, wherein the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, and sulfonamide; R2 is selected from substituted or unsubstituted C1-C10 straight-chain or branched alkyl, phenyl, thiazolyl, imidazolyl, furanyl, and the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, and sulfonamide. Ring A is selected from substituted or unsubstituted azaaryl groups, and the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, and sulfonamide. R3 is an azide group, alkynyl group, -NHR9 group, sulfonic acid group, or phosphoric acid group, wherein R9 is H or a C1-C6 straight-chain or branched alkyl group; R4 is an alkyl sulfonate anion or an alkyl phosphate anion; Y - It is an anion.

[0008] One embodiment of the present invention also provides a detection reagent, including a dye reagent, said dye reagent comprising one or more fluorescent compounds described in the foregoing embodiments.

[0009] One embodiment of the present invention also provides a kit for blood cell analysis, comprising the detection reagents described in the foregoing embodiments.

[0010] One embodiment of the present invention also provides the application of the detection reagent or kit described in the foregoing embodiments in blood cell detection.

[0011] In some implementations, the application specifically includes the following steps: (1) Mix the blood sample and diluent, and react; (2) Add the dye reagent to the mixture from step (1) and react; (3) Detect the scattered light signal and fluorescence signal of the mixture obtained in step (2); (4) Classify and count reticulocytes in blood cells based on scattered light signals and fluorescence signals.

[0012] In some implementations, the volume ratio of blood sample, fluorescent compound, and diluent is 1:(3-10):(10-1000).

[0013] In some implementations, the volume ratio of blood sample, fluorescent compound, and diluent is 1:(4-6):(100-500).

[0014] In some implementations, the reaction temperature of step (1) is 25-45°C.

[0015] In some implementations, the reaction temperature of step (1) is 30-40°C.

[0016] In some implementations, the reaction time for step (1) is 3-30 seconds.

[0017] In some implementations, the reaction time for step (2) is 5-15 seconds.

[0018] In some implementations, the reaction temperature of step (2) is 25-45°C.

[0019] In some implementations, the reaction temperature of step (2) is 30-40°C.

[0020] In some implementations, the reaction time for step (2) is 2-10 seconds.

[0021] In some implementations, the reaction time for step (2) is 3-8 seconds.

[0022] Compared with the prior art, the present invention has the following significant advantages: (1) Significantly enhanced fluorescence performance: By replacing the sulfur (S) atom in the benzothiazole ring of the existing technology with silicon (Si) atoms, the coplanarity and rigidity of the entire fluorophore molecule are effectively increased. This reduces the energy nonradiative decay caused by intramolecular vibration and rotation, thereby significantly improving the fluorescence quantum yield, resulting in higher and more stable emission fluorescence intensity. This significantly improves the detection sensitivity and signal-to-noise ratio, which is beneficial for ensuring the effective differentiation of reticulocytes at different maturity stages.

[0023] (2) Excellent staining specificity and selectivity: The hydrophobic fatty chain of the benzo[a]cyclic side chain is replaced with a hydrophilic group, which enhances the hydrophilicity of the dye molecule. This effectively reduces its non-specific binding to hydrophobic structures such as cell membrane lipids and intracellular proteins, enabling the dye to more specifically target and bind to nucleic acid substances in reticulocytes, reducing background interference and ensuring the accuracy and reliability of the counting results.

[0024] (3) Good compatibility in aqueous solution: The introduction of hydrophilic groups significantly improves the solubility of dyes in aqueous buffer solution, prevents the formation of hydrophobic aggregates, ensures the uniformity of the dyeing system, is more suitable for automated instrument detection environment, and has better test repeatability.

[0025] (4) The test results are more accurate after the sample is stored: The newly designed fluorescent dye molecules avoid the degradation region of RNA, effectively improving the problem of reduced reticulocyte ratio due to RNA degradation after clinical blood sample storage, and greatly improving the redundancy of operation time for clinical testing.

[0026] (5) High sensitivity: After the sample is treated with auxiliary diluent, the distinction between reticulocytes and mature red blood cells is increased, and the binding specificity of reticulocytes and fluorescent dyes is enhanced, which effectively improves the sensitivity of reticulocyte detection. Attached Figure Description

[0027] Figure 1 This is a scatter plot of cell classification obtained from sample 1-1 in Example 10 of the present invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity.

[0028] Figure 2 This is a scatter plot of cell classification obtained from sample 2-1 in Example 11 of the present invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity.

[0029] Figure 3 This is a scatter plot of cell classification obtained from sample 3-1 in Example 12 of this invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity.

[0030] Figure 4 The image shows a scatter plot of cell classification obtained from sample 1-1 in Comparative Example 1 of this invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity.

[0031] Figure 5 The image shows a scatter plot of cell classification obtained from sample 1-1 in Comparative Example 2 of this invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity.

[0032] Figure 6 This is a comparison graph of reticulocyte count in Example 11 and reticulocyte count using the standard method, where the X-axis represents the reticulocyte count result using the standard method and the Y-axis represents the reticulocyte count result in Example 11. Detailed Implementation

[0033] definition Unless otherwise stated, the terms used herein have the following meanings.

[0034] As used herein, the terms "aromatic ring" and "aryl" refer to a monocyclic or polycyclic aromatic ring having 3 to 20 carbon atoms, optionally also containing 1 to 3 heteroatoms selected from N, O, and S. Preferably, the term "aromatic ring" as used in this invention refers to an aromatic ring having at least 6 ring carbon atoms. More preferably, "aromatic ring" is a benzene ring or a polycyclic aryl group, and more preferably, a naphthalene ring, anthracene ring, or biphenyl.

[0035] As used herein, the term "alkyl," whether used alone or in combination with other groups, refers to straight-chain alkyl and branched alkyl groups containing 1-30, preferably 1-12, more preferably 1-8, and most preferably 1-6 carbon atoms. When referring to a single straight-chain alkyl group such as "n-propyl," it specifically refers to a straight-chain alkyl group; when referring to a single branched alkyl group such as "isopropyl," it specifically refers to a branched alkyl group. For example, "C1-6 alkyl" includes C1-4 alkyl, C1-3 alkyl, methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Similar rules apply to other groups used in this specification.

[0036] As used herein, the term "alkoxy" refers to an alkyl group as defined above that contains an oxygen atom, which is bonded to the remainder of the parent molecule via the oxygen atom.

[0037] As used herein, the term "alkenyl" or "chain alkenyl" refers to a straight or branched carbon chain containing one or more unsaturated double bonds in its molecular chain and comprising 2-30, preferably 6-14, more preferably 2-4 carbon atoms.

[0038] As used herein, the term "alkynyl" refers to a straight or branched carbon chain containing one or more unsaturated triple bonds in its molecular chain and comprising 2-30, preferably 6-14, more preferably 2-4 carbon atoms.

[0039] The term "halogen" as used in this article includes fluorine, chlorine, bromine, and iodine.

[0040] As used herein, the term "sulfonic acid group" refers to either the -SO3H group or the -SO3-M group, where M is the opposite ion, including, for example, an alkali metal ion (e.g., K+). + (Ions) or alkaline earth metal ions.

[0041] The term "biological sample" as used in this article includes, but is not limited to, peptides, proteins, nucleic acids, and cellular nucleic acid material (including DNA, RNA, and organelles containing DNA and RNA) in blood.

[0042] The compounds disclosed in this invention This invention provides a fluorescent compound capable of binding to intracellular nucleic acid substances (including DNA, RNA, and organelles containing DNA and RNA). The fluorescent compound emits fluorescence upon irradiation with excitation light of a specific wavelength. The fluorescent compound, which can be used in this invention, has any one of the following structural formulas I to IV: ; In structural formulas I to IV, X is selected from any one of Si(CH3)2, NH, and N(CH3); m is an integer from 1 to 6; R1 is selected from substituted or unsubstituted benzene, biphenyl, monocyclic aryl, polycyclic aryl, monocyclic heteroaryl, polycyclic heteroaryl, H, -NHR5, -NR6R7, wherein R5, R7 and R8 are each independently selected from H or C1-C8 straight-chain or branched alkyl, wherein the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, sulfonamide; R2 is selected from substituted or unsubstituted C1-C10 straight-chain or branched alkyl, phenyl, thiazolyl, imidazolyl, furanyl, and the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, and sulfonamide. Ring A is selected from substituted or unsubstituted azaaryl groups, and the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, and sulfonamide. R3 is an azide group, alkynyl group, -NHR9 group, sulfonic acid group, or phosphoric acid group, wherein R9 is H or a C1-C6 straight-chain or branched alkyl group; R4 is an alkyl sulfonate anion or an alkyl phosphate anion; Y - It is an anion.

[0043] In some implementation schemes, R2 is selected from any one of the following groups, whether substituted or unsubstituted: oxacyclopropane, azircyclopropane, thiocyclopropane, oxacyclobutane, azircyclobutane, thiocyclobutane, tetrahydrofuranyl, dioxacycloyl, tetrahydropyrrolyl, piperazinyl, tetrahydrothiopheneyl, hexahydropyrazinyl, TMP-yl, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, pyrrolyl, indolyl, furanyl, thiopheneyl, pyridinyl, DMAP-yl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, pyrazolyl, imidazolyl, benzofuranyl, benzothiopheneyl, piperazinyl, CDI-yl, quinolinyl, isoquinolinyl, pyrimidinyl, pyranoneyl, 1,2,3-triazolyl, 1,2,4-triazolyl, purinyl, and HOBt-yl.

[0044] In some implementation schemes, Ring A is selected from any one of the following: pyrrole ring, imidazole ring, pyrazole ring, thiazole ring, isothiazolyl, oxazole ring, isoxazole ring, 1,3,4-thiadiazolyl, triazole ring, tetrazolyl ring, pyridine ring, pyridazine ring, pyrimidine ring, pyrazine ring, triazine ring, tetraazine ring, indole ring, quinoline ring, isoquinoline ring, quinoxaline ring, quinazoline ring, purine ring, benzimidazole ring, acridine ring, phenazine ring, and carbazole ring.

[0045] In some embodiments, R1 is selected from substituted or unsubstituted phenyl, naphthyl, anthraceneyl, biphenyl, thiazolyl, thiophene, imidazolyl, thiazinyl, H, -NHR5, -NR6R7, wherein R5, R7 and R8 are each independently selected from H or C1-C8 straight-chain or branched alkyl, wherein the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicycline, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, and sulfonamide.

[0046] In some implementation schemes, Y - It includes halogen-based anions, sulfate-based anions, sulfonic acid-based anions, phosphate-based anions, and carboxylic acid-based anions.

[0047] In some implementation schemes, Y - For F - Cl - ,Br - I - NO2- NO3 - CH3SO3 - C2H5SO3 - C6H5SO3 - HSO3 - HSO4 - H2PO4 - HCOO - OH - CH3COO - C2H5COO - C6H5COO - BF4 - PF6 - SbF6 - ClO4 - Any one of them.

[0048] In some embodiments, the fluorescent compound has any one of the following structural formulas, wherein Y - For F - Cl - ,Br - I - NO2 - NO3 - CH3SO3 - C2H5SO3 - C6H5SO3 - HSO3 - HSO4 - H2PO4 - HCOO - OH - CH3COO - C2H5COO - C6H5COO - BF4 - PF6 - SbF6 - ClO4 - Any one of the following, where m is an integer from 1 to 6: Structural Formula 1; Structural Formula 2; Structure 3; Structural Formula 4; Structural formula 5; Structure 6; Structural formula 7; Structural formula 8; Structural formula 9; Structural formula 10; Structural formula 11; Structural formula 12; Structural formula 13; Structural formula 14; Structural formula 15; Structure 16; Structural formula 17; Structural Formula 18 Structural formula 19; Structural formula 20; Structural formula 21; Structural formula 22; Structural formula 23; Structural formula 24; Structural formula 25; Structural formula 26; Structural formula 27; Structural formula 28; Structural formula 29; Structural formula 30; Structural formula 31; Structural formula 32; Structural formula 33; Structural formula 34; Structural formula 35; Structural formula 36.

[0049] The compounds of one embodiment of the present invention can be used directly for staining biological samples as salts as described herein. Alternatively, in one embodiment, the compounds of one embodiment of the present invention can be used as derivatives of the compounds shown in structural formulas I to IV, and structural formulas 1 to 42, including but not limited to conjugates.

[0050] In one embodiment of the present invention, the compound itself has almost no fluorescence when no nucleic acid is present. After binding with nucleic acid to form a complex, the fluorescence intensity increases rapidly and the spectrum is in the near-infrared region, avoiding interference from background fluorescence and helping to improve the accuracy of detection results. It can be used for staining various biological samples on a flow cytometer.

[0051] Typically, conjugates are used in fluorescence activated cell sorting (FACS). As used herein, "conjugate" refers to a compound formed by covalently linking the compound of this invention with other molecules. Molecules that can be conjugated to the compound of this invention can be molecules that specifically bind to cells or cellular components, including but not limited to antibodies, antigens, receptors, ligands, enzymes, substrates, coenzymes, etc. Generally, the test sample is incubated with the conjugate for a period of time, allowing the conjugate to specifically bind to certain cells or cellular components in the test sample; this binding of the conjugate to cells or cellular components can also be referred to as staining. This staining step can be performed sequentially multiple times, or multiple staining steps can be performed simultaneously using multiple conjugates. After staining, the sample is analyzed in a fluorescence activated cell sorting system, where an excitation light source excites the compound of this invention in the conjugate, and an assay device measures the emitted light produced by the excited compound.

[0052] Alternatively, in another embodiment, the conjugate can also be used in solid-phase immunoassays, such as sandwich immunoassays. The techniques for solid-phase immunoassays are well known in the art and are available from standard textbooks. The conjugate of the present invention can be used as a variety of suitable components in solid-phase immunoassays.

[0053] The detection reagent of the present invention One embodiment of the present invention also provides a detection reagent for staining biological samples, comprising a dye reagent including one or more fluorescent compounds described in the foregoing embodiments.

[0054] In some implementations, the detection reagent also includes a diluent, which includes an alkaline buffer or a small molecule alcohol.

[0055] Small molecule alcohols The addition of small molecule alcohols to reticulocyte detection reagents is due to their amphiphilic nature, which allows them to selectively and gently act on the phospholipid bilayer of mature erythrocytes. By inserting themselves between membrane lipids, they weaken hydrophobic interactions and reduce membrane structural stability, thus achieving complete lysis of mature erythrocytes to eliminate detection interference without damaging target cells such as reticulocytes and leukocytes. At the same time, they can reduce the surface tension of the solution, regulate osmotic pressure, and protect cell morphology, providing a stable reaction environment for the staining, identification, and accurate counting of reticulocytes.

[0056] In some implementations, the small molecule alcohol is selected from methanol, ethanol, isopropanol, propanol, ethylene glycol, 1,2-propanediol, and 1,3-propanediol.

[0057] In some embodiments, the small molecule alcohol is selected from propanol and isopropanol.

[0058] In some embodiments, the concentration of the small molecule alcohol in the diluent is 3-20 v / v.

[0059] In some embodiments, the concentration of the small molecule alcohol in the diluent is 5-10 v / v.

[0060] Buffer system for maintaining pH 8-10 The detection reagents disclosed in this invention may optionally include a buffer system to maintain a stable pH value. The reticulocyte detection reagent uses an alkaline buffer system with a pH of 8-10, mainly because this range allows basic staining / fluorescent dyes to bind efficiently to the negatively charged RNA in reticulocytes, improving the staining signal and cell differentiation, while stabilizing cell morphology and preventing RNA degradation.

[0061] In some implementations, the buffer system used to maintain a pH of 8-10 is selected from Tris buffer system, AMP buffer system, and borate buffer system.

[0062] Other components The detection reagents disclosed in this invention may optionally contain surfactants, osmotic pressure regulators, preservatives, or anticoagulants.

[0063] Osmotic pressure regulator The detection reagents disclosed in this invention may optionally contain substances that regulate osmotic pressure, such as alkali metal salts, sugars, etc.

[0064] Generally speaking, excessively low osmotic pressure can also lead to excessive damage to leukocyte membranes. Therefore, the osmotic pressure of the detection reagent disclosed in this invention is typically maintained at 20-150 mOsm / kg.

[0065] Preservatives or anticoagulants The test reagents disclosed in this invention may optionally contain some conventional preservatives or anticoagulants to extend the shelf life of the reagents.

[0066] In some embodiments, the preservatives are preferably Kathon and methylparaben, used at a concentration of 0.05-1.5 g / L, preferably 0.05-0.5 g / L.

[0067] In some embodiments, the anticoagulant is selected from either EDTA-2Na or EDTA-2K.

[0068] In some embodiments, the concentration of the anticoagulant in the diluent is 0.05-0.5 g / L.

[0069] In some embodiments, the concentration of the anticoagulant in the diluent is 0.1-0.3 g / L.

[0070] The reagent kit of the present invention Another aspect of the present invention provides a kit comprising the detection reagents disclosed herein. The kit can be used to process blood samples, particularly those containing reticulocytes, identifying and counting the reticulocytes simultaneously.

[0071] In the kit, the components of the dye reagent disclosed in this invention can be presented as a single package, or the compounds of formulas I to IV (fluorescent compounds) can be packaged separately from other components, presenting as two or more separate packages.

[0072] Since the fluorescent compounds used in the detection reagents of this invention are more stable in non-aqueous solvents, it is preferable to store them separately from the water-soluble components of the dye reagents disclosed in this invention. It is preferable to store the fluorescent compounds in an organic solvent. For the organic solvent, there are no other special limitations as long as it can fully dissolve the fluorescent compounds and has a certain solubility in water; commonly used organic solvents such as methanol, ethanol, ethylene glycol, glycerol, and dimethyl sulfoxide are all acceptable.

[0073] The storage concentration of fluorescent compounds in organic solvents should be sufficient to ensure complete dissolution of the compound and not lower than its final usable concentration. Generally, the storage concentration range of fluorescent compounds in organic solvents is preferably from 0.01 ppm to 1000 ppm, and more preferably from 1 ppm to 100 ppm.

[0074] In this invention, the water-soluble component is referred to as the "diluent," and the component containing the fluorescent compound is referred to as the "dye reagent." When using the kit disclosed in this invention, the dye reagent is mixed with the diluent and blood sample at a certain volume ratio for a period of time before detection; alternatively, the dye reagent and diluent can be mixed first, and then mixed with the blood sample at a certain volume ratio before detection. There are no particular limitations on the volume ratio of the dye reagent to the diluent; it is typically mixed at a ratio of 1:10 to 1:100, preferably 1:40 to 1:60.

[0075] Application of the detection reagents or kits of the present invention This invention also discloses a method for classifying and counting reticulocytes, the method comprising the following steps: a. Mix the diluent disclosed in this invention with a blood sample, then add the dye reagent to the mixture and allow it to react. The fluorescent compound enters the cell and binds to nucleic acid substances; b. Detection of scattered light and fluorescence signals: After the product of the reaction is irradiated with excitation light of a certain wavelength, different levels of fluorescence signals will be generated according to the degree of binding of the fluorescent compound; different types of cells have different cell volumes, and the cell size will be changed to different degrees after reacting with the above reagents, which will produce scattered light signals of different magnitudes. c. Classify reticulocytes based on the differences between scattered light signals and fluorescence signals, and count the reticulocytes based on their count values.

[0076] The mixing of the dye reagent disclosed in this invention with a blood sample can be achieved by first pre-mixing the components of the dye reagent disclosed in this invention, and then mixing the mixture with the blood sample; alternatively, the components of the dye reagent disclosed in this invention can be added to the blood sample simultaneously and mixed together. The mixed sample is then reacted at a certain temperature for a period of time to dissolve and destroy red blood cells and platelets, facilitating the staining of reticulocytes. There are no particular limitations on the mixing ratio of the blood sample and the dye reagent disclosed in this invention, provided that the desired classification effect for different cell types is achieved.

[0077] In this invention, the volume ratio of blood sample to the fluorescent compound and diluent disclosed herein is 1:(3-10):(10-1000), preferably 1:(4-6):(100-500). After reagent treatment, leukocytes are slightly damaged, sufficient to allow the fluorescent compounds of structural formulas I to IV disclosed herein to enter the cell and bind to nucleic acid substances. The nucleic acid substances refer to DNA and RNA within the cell nucleus and organelles containing DNA or RNA.

[0078] Example The embodiments provided below are for further illustrative purposes only and are not intended to limit the scope of the specification in any way.

[0079] Unless otherwise stated, the reagent components used in the following examples are analytical grade, and the solvent used is water. The compounds of general formulas I to IV disclosed in this invention, which are used as fluorescent compounds, can first be prepared into a stock solution with an organic solvent. The blood cell detection equipment used is the CF9600[A1] fully automated blood cell analyzer manufactured by Shenzhen Coman Medical Equipment Co., Ltd., with an excitation wavelength of 635nm.

[0080] Example 1 Preparation of fluorescent compound 1 m is 5.

[0081] Preparation method of fluorescent compound 1: 20 mmol of 2,3,3-trimethyl-3H-naphtho[2,3-d][1,3]azasilane ring and 22 mmol of 5-bromopentane-1-sulfonate were added to a 50 mL round-bottom flask containing 20 mL of toluene under argon protection. The reaction was heated to reflux for 18 hours and then stopped. After cooling, the mixture was filtered to obtain a precipitate, and the filter cake was washed with diethyl ether. After drying, a yellow solid powder was obtained, with a crude yield of 50%.

[0082] In 60 ml of acetic acid, 10 mmol of 5-(2,3,3-trimethyl-3H-naphtho[2,3-d][1,3]azasilane-1-yl)pentane-1-sulfonate and 30 mmol of N,N'-diphenylmethanemid were heated and stirred in an oil bath at 90 °C for 1.5 h. The resulting red oily substance was washed three times with petroleum ether to remove acetic acid. Then, a certain amount of diethyl ether was added to precipitate an orange solid powder, which was filtered and dried. The crude product was separated by silica gel column chromatography, and the yellow fraction was collected using dichloromethane:methanol = 100:3 as the eluent, with a yield of 43%.

[0083] 10 mmol of 4-(4-methylpyridin-1(4H)-yl)morpholine and 10 mL of pyridine were added to the mixture, and the mixture was heated and stirred in an oil bath at 90 °C for 1.5 hours. The reaction solution was poured into diethyl ether, and dark black particles precipitated. The precipitate was filtered and dried. The dye was separated by silica gel column chromatography, and the blue fraction was collected using dichloromethane:methanol = 100:5 as the eluent to give fluorescent compound 1, with a yield of 50%.

[0084] Structure confirmation of fluorescent compound 1: 1H-NMR δ (400 MHz, CD3OD, TMS) 0.65(s,6H),1.29-1.32(q,8H),3.43(t,4H),3.79(t,4H),4.08(m,2H),5.24(d,1H ),6.34(d,2H),6.55(d,1H),6.68(s,1H),7.54(m,2H),7.81-7.86(m,4H),8.60(d, 2H). M.S. (EI) C 30 H 37 N3O4SSi: m / z: 563.23 [M] + .

[0085] Example 2 Preparation of fluorescent compound 2 m is 5.

[0086] Preparation method of fluorescent compound 2: 20 mmol of 2-methyl-1H-naphtho[2,3-d]imidazole and 22 mmol of 5-bromopentane-1-sulfonate were added to a 50 mL round-bottom flask containing 20 mL of toluene under argon protection. The reaction was heated to reflux for 18 hours and then stopped. After cooling, the mixture was filtered to obtain a precipitate, and the filter cake was washed with diethyl ether. After drying, a pale pink solid powder was obtained, with a crude yield of 54%.

[0087] In 60 mL of acetic acid, 10 mmol of 5-(2-methyl-1H-naphtho[2,3-d]imidazol-3-onthiol-3-yl)pentane-1-sulfonate and 30 mmol of N,N'-diphenylmethanemid were heated and stirred in an oil bath at 90 °C for 1.5 h. The resulting red oily substance was washed three times with petroleum ether to remove acetic acid. Then, a certain amount of diethyl ether was added to precipitate a pink solid powder, which was filtered and dried. The crude product was separated by silica gel column chromatography, and the yellow fraction was collected using dichloromethane:methanol = 100:3 as the eluent, with a yield of 51%.

[0088] 10 mmol of 4-(4-methylpyridin-1(4H)-yl)morpholine and 10 mL of pyridine were added to the mixture, and the mixture was heated and stirred in an oil bath at 90 °C for 1.5 hours. The reaction solution was poured into diethyl ether, and dark black particles precipitated. The precipitate was filtered and dried. The dye was separated by silica gel column chromatography, and the blue fraction was collected using dichloromethane:methanol = 100:5 as the eluent to give fluorescent compound 2, with a yield of 59%.

[0089] Structure confirmation of fluorescent compound 2: 1H-NMR δ (400 MHz, CD3OD, TMS) 1.29-1.33(m,6H),2.11(m,2H),3.5(t,4H),3.85(t,4H),5.11(t,2H),6 .35(d,2H),6.56(s,1H),6.68(d,1H),6.71(d,1H),7.48(m,2H),7.69(s, 2H),7.84(m,2H),8.6(d,2H). M.S. (EI) C 28 H 32 N4O4S: m / z: 520.21 [M] + .

[0090] Example 3 Preparation of fluorescent compound 3 m is 5, Y - For Cl - .

[0091] Preparation method of fluorescent compound 3: 20 mmol of 1-methyl-1H-naphtho[2,3-d]imidazole and 22 mmol of 5-chloropentane-1-sulfonic acid were added to a 50 mL round-bottom flask containing 20 mL of toluene under argon protection. The reaction was heated to reflux for 18 hours and then stopped. After cooling, the mixture was filtered to obtain a precipitate, and the filter cake was washed with diethyl ether. After drying, a yellowish-brown solid powder was obtained, with a crude yield of 46%.

[0092] In 60 ml of acetic acid, 10 mmol of 1,2-dimethyl-3-(5-sulfopentyl)-1H-naphtho[2,3-d]imidazol-3-onium chloride and 30 mmol of N,N'-diphenylmethanemid were heated and stirred in an oil bath at 90 °C for 1.5 h. The resulting red oily substance was washed three times with petroleum ether to remove acetic acid. Then, a certain amount of diethyl ether was added to precipitate a pink solid powder, which was filtered and dried. The crude product was separated by silica gel column chromatography, using dichloromethane:methanol = 100:3 as the eluent, and the pale yellow fraction was collected, with a yield of 41%.

[0093] 10 mmol of 4-(4-methylpyridin-1(4H)-yl)morpholine and 10 mL of pyridine were added, and the mixture was heated and stirred in an oil bath at 90 °C for 1.5 hours. The reaction solution was poured into diethyl ether, and dark black particles precipitated. The precipitate was filtered and dried. The dye was separated by silica gel column chromatography, and the blue fraction was collected using dichloromethane:methanol = 100:5 as the eluent to give fluorescent compound 3, with a yield of 44%.

[0094] Structure confirmation of fluorescent compound 3: 1 H-NMR δ (400 MHz, CD3OD, TMS) 1.32(m,2H),1.47(m,2H),2.11(m,2H),3.01(t,2H),3.42(t,4H),3.76(s,3H),3.89(t,4H),5. 11(t,2H),6.27(d,2H),6.54-6.71(m,3H),7.48(m,2H),7.69(s,2H),7.84(m,2H),8.69(d,2H). M.S. (EI) C 29 H 35 N4O4S + m / z: 535.24 [M-Cl] + .

[0095] Example 4 Preparation of fluorescent compound 5 m is 5, Y - For Cl - .

[0096] Preparation method of fluorescent compound 5: 20 mmol of 2,3,3-trimethyl-3H-naphtho[2,3-d][1,3]azasilane and 22 mmol of 5-chloropentane-1-sulfonic acid were added to a 50 mL round-bottom flask containing 20 mL of toluene under argon protection. The reaction was heated to reflux for 18 hours and then stopped. After cooling, the mixture was filtered to obtain a precipitate, and the filter cake was washed with diethyl ether. After drying, a yellowish-brown solid powder was obtained, with a crude yield of 56%.

[0097] In 60 ml of acetic acid, 10 mmol of 2,3,3-trimethyl-1-(5-sulfopentyl)-3H-naphtho[2,3-d][1,3]azasilane-1-onium chloride and 30 mmol of N,N'-diphenylmethanemid were heated and stirred in an oil bath at 90 °C for 1.5 h. The resulting red oily substance was washed three times with petroleum ether to remove acetic acid. Then, a certain amount of diethyl ether was added to precipitate a pink solid powder, which was filtered and dried. The crude product was separated by silica gel column chromatography, and the yellow fraction was collected using dichloromethane:methanol = 100:3 as the eluent, with a yield of 45%.

[0098] 10 mmol of 7-methyl-7H-thiazo[3,2-a]pyridine and 10 mL of pyridine were added to the mixture, and the mixture was heated and stirred in an oil bath at 90 °C for 1.5 hours. The reaction solution was poured into diethyl ether, and dark purple particles precipitated. The precipitate was filtered and dried. The dye was separated by silica gel column chromatography, and the blue fraction was collected using dichloromethane:methanol = 100:5 as the eluent to give fluorescent compound 5, with a yield of 47%.

[0099] Structure confirmation of fluorescent compound 5: 1 H-NMR δ (400 MHz, CD3OD, TMS) 0.66(s,6H),1.28-1.33(m,4H),1.48(m,2H),3.01(t,2H),4.01(t,2H),5.21(s,1H),5.31-5.37(q,2H),6. 28(d,1H),6.5(d,1H),6.57(s,1H),7.54(m,2H),7.81-7.84(m,4H),8.03(d,1H),8.69(d,2H),11.0(s,1H). MS(EI)C 28 H 31 N2O3S2Si + m / z: 535.15 [M-Cl] + .

[0100] Example 5 Preparation of fluorescent compound 7 m is 5, Y - For Cl - .

[0101] Preparation method of fluorescent compound 7: 20 mmol of 2,3,3-trimethyl-3H-naphtho[2,3-d][1,3]azasilane and 22 mmol of 5-chloropentane-1-sulfonic acid were added to a 50 mL round-bottom flask containing 20 mL of toluene under argon protection. The reaction was heated to reflux for 18 hours and then stopped. After cooling, the mixture was filtered to obtain a precipitate, and the filter cake was washed with diethyl ether. After drying, a yellowish-brown solid powder was obtained, with a crude yield of 49%.

[0102] In 60 ml of acetic acid, 10 mmol of 2,3,3-trimethyl-1-(5-sulfopentyl)-3H-naphtho[2,3-d][1,3]azasilane-1-onium chloride and 30 mmol of N,N'-diphenylmethanemid were heated and stirred in an oil bath at 90 °C for 1.5 h. The resulting red oily substance was washed three times with petroleum ether to remove acetic acid. Then, a certain amount of diethyl ether was added to precipitate a pink solid powder, which was filtered and dried. The crude product was separated by silica gel column chromatography, and the yellow fraction was collected using dichloromethane:methanol = 100:3 as the eluent, with a yield of 45%.

[0103] 10 mmol of 7-methyl-7H-oxazolo[3,2-a]pyridine and 10 mL of pyridine were added to the mixture, and the mixture was heated and stirred in an oil bath at 90 °C for 1.5 hours. The reaction solution was poured into diethyl ether, and dark purple particles precipitated. The precipitate was filtered and dried. The dye was separated by silica gel column chromatography, and the blue fraction was collected using dichloromethane:methanol = 100:5 as the eluent to give fluorescent compound 7, with a yield of 43%.

[0104] The structure of fluorescent compound 7 was confirmed: 1 H-NMR δ (400 MHz, CD3OD, TMS) 0.66(s,6H),1.28-1.33(m,4H),1.5(m,2H),3.01(t,2H),4.11(t,2H),4.93(s,1H),5.3(d,1H),5.4-5.47 (q,2H),6.52(d,1H),6.67(s,1H),7.52(m,2H),7.78-7.84(m,4H),8.03(d,1H),8.5(d,2H),11.0(s,1H). MS(EI)C 28 H 31 N2O4SSi + m / z: 519.18[M-Cl] + .

[0105] Example 6 Preparation of fluorescent compound 8 m is 5, Y - For Cl - .

[0106] Preparation method of fluorescent compound 8: 20 mmol of 2-methyl-1H-naphtho[2,3-d]imidazole and 22 mmol of 5-chloropentane-1-sulfonic acid were added to a 50 mL round-bottom flask containing 20 mL of toluene under argon protection. The reaction was heated to reflux for 18 hours and then stopped. After cooling, the mixture was filtered to obtain a precipitate, and the filter cake was washed with diethyl ether. After drying, a brown solid powder was obtained, with a crude yield of 54%.

[0107] In 60 ml of acetic acid, 10 mmol of 3-(5-sulfopentyl)-1H-naphtho[2,3-d]imidazol-3-onium chloride and 30 mmol of N,N'-diphenylmethanemid were heated and stirred in an oil bath at 90 °C for 1.5 h. The resulting red oily substance was washed three times with petroleum ether to remove acetic acid. Then, a certain amount of diethyl ether was added to precipitate a pink solid powder, which was filtered and dried. The crude product was separated by silica gel column chromatography, and the yellow fraction was collected using dichloromethane:methanol = 100:3 as the eluent, with a yield of 43%.

[0108] 10 mmol of 7-methyl-7H-thiazo[3,2-a]pyridine and 10 mL of pyridine were added to the mixture, and the mixture was heated and stirred in an oil bath at 90 °C for 1.5 hours. The reaction solution was poured into diethyl ether, and dark purple particles precipitated. The precipitate was filtered and dried. The dye was separated by silica gel column chromatography, and the blue fraction was collected using dichloromethane:methanol = 100:5 as the eluent to give fluorescent compound 8, with a yield of 51%.

[0109] Structure confirmation of fluorescent compound 8: 1 H-NMR δ (400 MHz, CD3OD, TMS) 1.33(m,4H),1.49(m,2H),2.01(m,2H),3.01(t,2H),4.01(t,2H),5.01(t,2H),5.24(s,1H),5.47(d,1H),6. 29(d,1H),6.58-6.71(m,3H),7.46(m,2H),7.69(s,2H),7.84(m,2H),8.09(d,1H),8.49(d,2H),11.0(s,1H). MS(EI)C 26 H 26 N3O3S2 + m / z: 492.14 [M-Cl] + .

[0110] Example 7 Preparation of fluorescent compound 9 m is 5, Y - For Cl - .

[0111] Preparation method of fluorescent compound 9: 20 mmol of 1-methyl-1H-naphtho[2,3-d]imidazole and 22 mmol of 5-chloropentane-1-sulfonic acid were added to a 50 mL round-bottom flask containing 20 mL of toluene under argon protection. The reaction was heated to reflux for 18 hours and then stopped. After cooling, the mixture was filtered to obtain a precipitate, and the filter cake was washed with diethyl ether. After drying, a yellowish-brown solid powder was obtained, with a crude yield of 46%.

[0112] In 60 ml of acetic acid, 10 mmol of 1,2-dimethyl-3-(5-sulfopentyl)-3H-naphtho[2,3-d]imidazol-3-onium chloride and 30 mmol of N,N'-diphenylmethanemid were heated and stirred in an oil bath at 90 °C for 1.5 hours. The resulting red oily substance was washed three times with petroleum ether to remove acetic acid. Then, a certain amount of diethyl ether was added to precipitate a pink solid powder, which was filtered and dried. The crude product was separated by silica gel column chromatography, using dichloromethane:methanol = 100:3 as the eluent, and the pale yellow fraction was collected, with a yield of 41%.

[0113] 10 mmol of 7-methyl-7H-thiazo[3,2-a]pyridine and 10 mL of pyridine were added to the mixture, and the mixture was heated and stirred in an oil bath at 90 °C for 1.5 hours. The reaction solution was poured into diethyl ether, and dark blue particles precipitated. The precipitate was filtered and dried. The dye was separated by silica gel column chromatography, and the blue fraction was collected using dichloromethane:methanol = 100:5 as the eluent to give fluorescent compound 9, with a yield of 44%.

[0114] Structure confirmation of fluorescent compound 9: 1 H-NMR δ (400 MHz, CD3OD, TMS): 1.33(m,2H),1.51(m,2H),2.1(m,2H),3.01(t,2H),3.76(s,3H),5.01(t,2H),5.23(s,1H),5.37(d,1H),6.2 9(s,1H),6.58(d,1H),6.71(d,1H),7.46(m,2H),7.64(s,2H),7.84(m,2H),8.03(d,1H),8.62(d,2H),11.0(s,1H). MS(EI)C 27 H 28 N3O3S2 + m / z: 506.16 [M-Cl] + .

[0115] Example 8 Preparation of fluorescent compound 10 m is 5.

[0116] Preparation method of fluorescent compound 10: 20 mmol of 2,3,3-trimethyl-3H-naphtho[2,3-d][1,3]azasilane ring and 22 mmol of 5-bromopentane-1-sulfonate were added to a 50 mL round-bottom flask containing 20 mL of toluene under argon protection. The reaction was heated to reflux for 18 hours and then stopped. After cooling, the mixture was filtered to obtain a precipitate, and the filter cake was washed with diethyl ether. After drying, a yellow solid powder was obtained, with a crude yield of 50%.

[0117] In 60 ml of acetic acid, 10 mmol of 5-(2,3,3-trimethyl-3H-naphtho[2,3-d][1,3]azasilane-1-yl)pentane-1-sulfonate and 30 mmol of N,N'-diphenylmethanemid were heated and stirred in an oil bath at 90 °C for 1.5 h. The resulting red oily substance was washed three times with petroleum ether to remove acetic acid. Then, a certain amount of diethyl ether was added to precipitate an orange solid powder, which was filtered and dried. The crude product was separated by silica gel column chromatography, and the yellow fraction was collected using dichloromethane:methanol = 100:3 as the eluent, with a yield of 43%.

[0118] 10 mmol of 7-methyl-7H-thiazo[3,2-a]pyridine and 10 mL of pyridine were added to the mixture, and the mixture was heated and stirred in an oil bath at 90 °C for 1.5 hours. The reaction solution was poured into diethyl ether, and dark blue particles precipitated. The precipitate was filtered and dried. The dye was separated by silica gel column chromatography, and the blue fraction was collected using dichloromethane:methanol = 100:5 as the eluent to give fluorescent compound 10, with a yield of 52%.

[0119] Structure confirmation of fluorescent compound 10: 1 H-NMR δ (400 MHz, CD3OD, TMS) 0.66(s,6H),1.29-1.33(m,8H),4.01(m,2H),5.21(s,1H),5.34(d,1H),5.37(d,1H),6.3(d ,1H),6.52(d,1H),6.58(d,1H),7.56(m,2H),7.78-7.84(m,4H),8.03(d,1H),8.59(d,2H). M.S. (EI) C 28 H 30 N₂O₃S₂Si: m / z: 534.15 [M] + .

[0120] Example 9 Preparation of fluorescent compound 11 m is 5.

[0121] Preparation method of fluorescent compound 11: 20 mmol of 2-methyl-1H-naphtho[2,3-d]imidazole and 22 mmol of 5-bromopentane-1-sulfonate were added to a 50 mL round-bottom flask containing 20 mL of toluene under argon protection. The reaction was heated to reflux for 18 hours and then stopped. After cooling, the mixture was filtered to obtain a precipitate, and the filter cake was washed with diethyl ether. After drying, a pale pink solid powder was obtained, with a crude yield of 54%.

[0122] In 60 mL of acetic acid, 10 mmol of 5-(2-methyl-1H-naphtho[2,3-d]imidazol-3-onthiol-3-yl)pentane-1-sulfonate and 30 mmol of N,N'-diphenylmethanemid were heated and stirred in an oil bath at 90 °C for 1.5 h. The resulting red oily substance was washed three times with petroleum ether to remove acetic acid. Then, a certain amount of diethyl ether was added to precipitate a pink solid powder, which was filtered and dried. The crude product was separated by silica gel column chromatography, and the yellow fraction was collected using dichloromethane:methanol = 100:3 as the eluent, with a yield of 51%.

[0123] 10 mmol of 7-methyl-7H-thiazo[3,2-a]pyridine and 10 mL of pyridine were added to the mixture, and the mixture was heated and stirred in an oil bath at 90 °C for 1.5 hours. The reaction solution was poured into diethyl ether, and dark blue particles precipitated. The precipitate was filtered and dried. The dye was separated by silica gel column chromatography, and the blue fraction was collected using dichloromethane:methanol = 100:5 as the eluent to give fluorescent compound 11, with a yield of 57%.

[0124] Structural confirmation of fluorescent compound 11: 1 H-NMR δ (400 MHz, CD3OD, TMS) 1.3-1.33(m,6H),2.11(m,2H),5.01(t,2H),5.21(s,1H),5.38(d,1H),6.29(d,1H),6.58(s,1H ),6.65(d,1H),6.72(d,1H),7.48(m,2H),7.65(s,2H),7.84(m,2H),8.03(d,1H),8.69(d,2H). MS(EI)C 26 H 25 N3O3S2:m / z:491.13[M] + .

[0125] Example 10 The reagent kit of Example 10 of the present invention was prepared using the following components. Ten blood samples (5 μL each) were mixed with 1 mL of diluent to obtain cell suspensions, incubated at 37°C for 10 seconds, and then 20 μL of fluorescent dye stock solution was added, followed by incubation at 37°C for 5 seconds. Reticulocytes in the blood samples were detected using laser flow cytometry (excitation source: semiconductor laser, excitation wavelength: 635 nm). Forward scattering information at 1-10 degrees and lateral fluorescence information at 90 degrees were detected. Based on the comprehensive analysis of the forward scattering and lateral fluorescence information, the scatter plot results for sample 1-1 are shown below. Figure 1 As shown.

[0126] Using the test reagents in Example 10, 10 blood samples were repeatedly tested. The same specimen was compared using the standard method recommended by the International Committee for Standardization of Hematology (ICSH). The test results for resistance to sample storage aging (after being placed at room temperature for 48 hours) are as follows: Example 11 The reagent kit of Example 11 of the present invention was prepared using the following components. Ten blood samples (5 μL each) were mixed with 1 mL of diluent to obtain cell suspensions, incubated at 37°C for 12 seconds, and then 20 μL of fluorescent dye stock solution was added, followed by incubation at 37°C for 7 seconds. Reticulocytes in the blood samples were detected using laser flow cytometry (excitation source: semiconductor laser, excitation wavelength: 635 nm). Forward scattering information at 1-10 degrees and lateral fluorescence information at 90 degrees were detected. Based on the comprehensive analysis of the forward scattering and lateral fluorescence information, the scatter plot results for sample 2-1 are shown below. Figure 2 As shown.

[0127] Using the test reagents in Example 11, repeatability tests were performed on 10 blood samples. The same specimen was compared using the standard method recommended by the International Committee for Standardization of Hematology (ICSH). The test results for resistance to sample storage aging (after being left at room temperature for 48 hours) are as follows: Example 12 The reagent kit of Example 12 of the present invention was prepared using the following components. Ten blood samples (5 μL each) were mixed with 0.8 mL of diluent to obtain cell suspensions, incubated at 37°C for 15 seconds, and then 20 μL of fluorescent dye stock solution was added, followed by incubation at 37°C for 6 seconds. Reticulocytes in the blood samples were detected using laser flow cytometry (excitation source: semiconductor laser, excitation wavelength: 635 nm). Forward scattering information at 1-10 degrees and lateral fluorescence information at 90 degrees were detected. Based on the comprehensive analysis of the forward scattering and lateral fluorescence information, the scatter plot results for sample 3-1 are shown below. Figure 3 As shown.

[0128] Using the test reagents in Example 12, repeatability tests were performed on 10 blood samples. The same specimen was compared using the standard method recommended by the International Committee for Standardization of Hematology (ICSH). The test results for resistance to sample storage aging (after being left at room temperature for 48 hours) are as follows: Comparative Example 1 As a control of Example 10, a kit for blood cell analysis was prepared using the following components. This kit differs from the kit used in Example 10 only in that the fluorescent compound in the dye reagent is a fluorescent dye of formula (I) described in prior art document CN101344472A: .

[0129] Ten blood samples (5 μL each) were mixed with 1 mL of diluent to obtain cell suspensions, incubated at 37°C for 10 seconds, and then 20 μL of fluorescent dye stock solution was added, followed by incubation at 37°C for 5 seconds. Reticulocytes in the blood samples were detected using laser flow cytometry (excitation source: semiconductor laser, excitation wavelength: 635 nm). Forward scattering information at 1-10 degrees and lateral fluorescence information at 90 degrees were detected. Based on the comprehensive analysis of the forward scattering and lateral fluorescence information, the scatter plot results for sample 1-1 are shown below. Figure 4 As shown.

[0130] Using the test reagents in Comparative Example 1, 10 blood samples were repeatedly tested. The same specimen was compared using the standard method recommended by the International Committee for Standardization of Hematology (ICSH). The test results for resistance to sample storage aging (after being left at room temperature for 48 hours) are as follows: Comparative Example 2 As a control of Example 10, a kit for blood cell analysis was prepared using the following components. This kit differs from the kit used in Example 10 only in that the fluorescent compound in the dye reagent is pigment compound B as described in prior art document CN1154966A: .

[0131] Ten blood samples (5 μL each) were mixed with 1 mL of diluent to obtain cell suspensions, incubated at 37°C for 10 seconds, and then 20 μL of fluorescent dye stock solution was added, followed by incubation at 37°C for 5 seconds. Reticulocytes in the blood samples were detected using laser flow cytometry (excitation source: semiconductor laser, excitation wavelength: 635 nm). Forward scattering information at 1-10 degrees and lateral fluorescence information at 90 degrees were detected. Based on the comprehensive analysis of the forward scattering and lateral fluorescence information, the scatter plot results for sample 1-1 are shown below. Figure 5 As shown.

[0132] Using the test reagents in Comparative Example 2, 10 blood samples were repeatedly tested. The same specimen was compared using the standard method recommended by the International Committee for Standardization of Hematology (ICSH). The test results for resistance to sample storage aging (after being left at room temperature for 48 hours) are as follows: The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A fluorescent compound, characterized in that, The fluorescent compound has the following structural formula I: It has any one of the following structural formulas I to IV: (I) (II) (III) (IV); In structural formulas I to IV, X is selected from any one of Si(CH3)2, NH, and N(CH3); m is an integer from 1 to 6; R1 is selected from substituted or unsubstituted benzene, biphenyl, monocyclic aryl, polycyclic aryl, monocyclic heteroaryl, polycyclic heteroaryl, H, -NHR5, -NR6R7, wherein R5, R7 and R8 are each independently selected from H or C1-C8 straight-chain or branched alkyl, wherein the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, sulfonamide; R2 is selected from substituted or unsubstituted C1-C10 straight-chain or branched alkyl, phenyl, thiazolyl, imidazolyl, furanyl, and the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, and sulfonamide. Ring A is selected from substituted or unsubstituted azaaryl groups, and the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, and sulfonamide. R3 is an azide group, alkynyl group, -NHR9 group, sulfonic acid group, or phosphoric acid group, wherein R9 is an H group or a C1-C6 straight-chain or branched alkyl group; R4 is an alkyl sulfonate anion or an alkyl phosphate anion; Y - It is an anion.

2. The fluorescent compound according to claim 1, characterized in that, Optional R2 is selected from any one of the following groups, whether substituted or unsubstituted: oxetane, azirne, thiohexacyclopropane, oxetane, azirne, thiohexacyclopropane, tetrahydrofuranyl, dioxanecyclo, tetrahydropyrrolyl, piperazinyl, tetrahydrothiopheneyl, hexahydropyrazinyl, TMP-yl, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, pyrrolyl, indolyl, furanyl, thiopheneyl, pyridinyl, DMAP-yl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, pyrazolyl, imidazolyl, benzofuranyl, benzothiopheneyl, piperazinyl, CDI-yl, quinolinyl, isoquinolinyl, pyrimidinyl, pyranoneyl, 1,2,3-triazolyl, 1,2,4-triazolyl, purinyl, and HOBt-yl. Optional Ring A is selected from any one of the following: pyrrole ring, imidazole ring, pyrazole ring, thiazole ring, isothiazolyl, oxazole ring, isoxazole ring, 1,3,4-thiadiazolyl, triazole ring, tetrazolyl ring, pyridine ring, pyridazine ring, pyrimidine ring, pyrazine ring, triazine ring, tetraazine ring, indole ring, quinoline ring, isoquinoline ring, quinoxaline ring, quinazoline ring, purine ring, benzimidazole ring, acridine ring, phenazine ring, and carbazole ring.

3. The fluorescent compound according to claim 1, characterized in that, Optionally, R1 is selected from substituted or unsubstituted phenyl, naphthyl, anthraceneyl, biphenyl, thiazolyl, thiophene, imidazolyl, thiazinyl, H, -NHR5, -NR6R7, wherein R5, R7 and R8 are each independently selected from H or C1-C8 straight-chain or branched alkyl, wherein the substituent is selected from at least one of alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, mercapto, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, carboxyl, sulfonyl, thiooxy, sulfinyl, sulfonamide; Optional, Y - The preferred anions are halogen-based anions, sulfate-based anions, sulfonic acid-based anions, phosphate-based anions, and carboxylic acid-based anions. - For F - Cl - ,Br - I - NO2 - NO3 - CH3SO3 - C2H5SO3 - C6H5SO3 - HSO3 - HSO4 - H2PO4 - HCOO - OH - CH3COO - C2H5COO - C6H5COO - BF4 - PF6 - SbF6 - ClO4 - Any one of them.

4. The fluorescent compound according to claim 1, characterized in that, The fluorescent compound has any one of the following structural formulas, wherein Y - For F - Cl - ,Br - I - NO2 - NO3 - CH3SO3 - C2H5SO3 - C6H5SO3 - HSO3 - HSO4 - H2PO4 - HCOO - OH - CH3COO - C2H5COO - C6H5COO - BF4 - PF6 - SbF6 - ClO4 - Any one of the following, where m is an integer from 1 to 6: Structural Formula 1; Structural Formula 2; Structure 3; Structural Formula 4; Structural formula 5; Structure 6; Structural formula 7; Structural formula 8; Structural formula 9; Structural formula 10; Structural formula 11; Structural formula 12; Structural formula 13; Structural formula 14; Structural formula 15; Structure 16; Structural formula 17; Structural Formula 18 Structural formula 19; Structural formula 20; Structural formula 21; Structural formula 22; Structural formula 23; Structural formula 24; Structural formula 25; Structural formula 26; Structural formula 27; Structural formula 28; Structural formula 29; Structural formula 30; Structural formula 31; Structural formula 32; Structural formula 33; Structural formula 34; Structural formula 35; Structural formula 36.

5. A detection reagent, comprising a dye reagent, characterized in that, The dye reagent includes the fluorescent compound as described in any one of claims 1-4.

6. The detection reagent according to claim 5, characterized in that, The detection reagent also includes a diluent, which includes a buffer system for maintaining a pH of 8-10 and a small molecule alcohol. Preferably, the concentration of the small molecule alcohol in the diluent is 3-20 v / v%, more preferably, the concentration of the small molecule alcohol in the diluent is 5-10 v / v%. Preferably, the buffer system used to maintain a pH value of 8-10 is selected from the Tris buffer system, AMP buffer system, and borate buffer system; Preferably, the small molecule alcohol is selected from methanol, ethanol, isopropanol, propanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, etc. More preferably, the small molecule alcohol is selected from propanol and isopropanol.

7. A reagent kit for blood cell analysis, characterized in that, Includes the detection reagents as described in any one of claims 5-6.

8. The use of the detection reagent as described in any one of claims 5-6 or the kit as described in claim 7 in blood cell detection.

9. The application according to claim 8, characterized in that, Includes the following steps: (1) Mix the blood sample and diluent, and react; (2) Add the dye reagent to the mixture from step (1) and react; (3) Detect the scattered light signal and fluorescence signal of the mixture obtained in step (2); (4) Classify and count reticulocytes in blood cells based on scattered light signals and fluorescence signals.

10. The application according to claim 9, characterized in that, The volume ratio of blood sample, fluorescent compound, and diluent is 1:(3-10):(10-1000), preferably 1:(4-6):(100-500). Alternatively, the reaction temperature in step (1) is 25-45°C, preferably 30-40°C; Alternatively, the reaction time of step (1) is 3-30 seconds, preferably, the reaction time of step (2) is 5-15 seconds; Alternatively, the reaction temperature in step (2) is 25-45°C, preferably 30-40°C; Alternatively, the reaction time of step (2) is 2-10 seconds, preferably 3-8 seconds.

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