Cyanine dye, preparation method and application thereof, sample analysis method and analyzer

Abstract

Relates to a fluorescent dye, in particular to a cyanine dye and a preparation method and application thereof. Specifically, the invention provides a compound with a structure as shown in a general formula I or a hydrate, a solvate, a stereoisomer, a tautomer or a crystal form thereof, and R1, R2 and R3 are defined in the specification. The cyanine dye provided by the invention has the advantages of good stability, good biological penetrating power and the like. # imgabs0 #

Description

Cyanine dye, preparation method, use, sample analysis method and analyzer thereof Technical Field

[0001] The present application relates to a fluorescent dye and its application, in particular to a cyanine dye, its preparation method and application, a sample analysis method and a sample analyzer. The present application also relates to a conjugate containing the cyanine dye and a composition for staining biological samples. Background Art

[0002] DNA (deoxyribonucleic acid) is a biological macromolecule that carries genetic information. Normal cells typically maintain a relatively stable diploid DNA content, but abnormal changes occur when disease occurs. Furthermore, DNA content generally varies between different organisms. Therefore, the specific identification and precise measurement of DNA, especially within living cells, is of great significance. The use of fluorescent dyes for qualitative or quantitative DNA analysis has attracted widespread interest among researchers due to their high sensitivity and rapid response.

[0003] Currently, commercial dyes mainly include phenanthridines, acridines, imidazoles, and cyanine families. However, the application process of these dyes has certain limitations. First, a large part of the dyes show fluorescence quenching after binding to DNA, resulting in low practical value in visualization applications such as fluorescence imaging. Second, the specificity of most current dyes is poor and they cannot specifically bind to DNA, which greatly limits their application potential. Third, most current dyes are highly toxic and carcinogenic in themselves, and require increasing the permeability of the cell membrane to label biological samples. However, this permeability treatment causes damage to cells and biological tissues, limiting their application in living cells.

[0004] Among the numerous fluorescent dyes, cyanine fluorescent dyes have been widely used as fluorescent probes for biomolecules due to their wide wavelength range, large molar extinction coefficient, and moderate fluorescence quantum yield. Although some cyanine dyes have been commercialized, most of these dyes are large, complex, and have low permeability to living cells. Furthermore, most dyes exhibit a small Stoke's shift, leading to significant crosstalk between the excitation and emission spectra, causing background interference and fluorescence self-quenching, which limits their application.

[0005] Microorganisms are tiny, simple organisms found in nature that are invisible to the naked eye and require magnification with specialized instruments. They include prokaryotes (such as bacteria, actinomycetes, mycoplasmas, rickettsiae, chlamydiae, and spirochetes), eukaryotes (such as fungi, protozoa, and algae), and non-cellular organisms (viruses and prions).

[0006] Many diseases are related to microorganisms. Diseases caused by pathogenic microorganisms include bacterial, viral, fungal, and protozoal diseases. Pathogenic microorganisms can cause blood-borne infections, including bacteremia, sepsis, toxemia, and septicemia. Sepsis, a systemic infectious syndrome (SIRS), can cause septic shock and multiple organ dysfunction, with a mortality rate as high as 30-50%. Therefore, rapid detection of microorganisms is of great significance.

[0007] Currently, the gold standard for clinical microbial diagnosis is blood culture. However, the entire process from blood culture to plate culture to results is cumbersome and time-consuming (typically over 3-5 days). By the time results are available, the patient's condition is often very serious. To address this, various new and effective detection methods have emerged, including immunoassays and electrochemical assays.

[0008] Immunoassays utilize antigen-antibody specific reactions to detect specific microorganisms. These include traditional lectin and precipitation assays, as well as newer enzyme-linked immunosorbent assays (ELISAs), radiolabeled assays, and chemiluminescent immunoassays. Immunoassays offer exceptional specificity and are faster than traditional culture methods. However, due to drawbacks such as difficulty obtaining antibodies, high antibody costs, insufficient sensitivity, and environmental susceptibility to reactivity, immunoassays have been hindered from widespread application in clinical microbiology testing.

[0009] Electrochemical detection primarily utilizes changes in electrical signals generated by microbial metabolism to detect microorganisms. Common methods include impedance analysis, potentiometry, and amperometry. Electrochemical detection equipment is small and low-cost, but lacks sensitivity.

[0010] Hemocyte-borne microorganisms are parasites that live in the blood or blood cells of animals, such as malaria and Toxoplasma gondii. Blood parasites have a shorter lifespan and are more harmful, so early and accurate diagnosis of blood parasites is essential for effective disease management and monitoring.

[0011] There are currently three main methods for detecting blood cell parasitic microorganisms. The first method is to accurately detect and quantify blood cell parasitic microorganisms by microscopic examination of blood smears, but this is highly dependent on the training and skills of the operator. The second method is a rapid diagnostic test (RDT) based on antigen-antibody reaction. This method does not require too high operating skills, but is usually more expensive and lacks sufficient sensitivity for low-level blood cell parasites. The third method is to detect parasites in blood cells when performing routine blood screening using a blood cell analyzer. This method is simple and easy to operate, has low cost and does not rely on the training and skills of the operator.

[0012] However, the accuracy of detecting parasites in the blood using hematology analyzers is still not high.

[0013] Summary of the Invention

[0014] In order to solve the above problems, the inventors of the present application have obtained new benzothiazole cyanine dyes through molecular modification, which have significantly improved thermal stability. Moreover, compared with existing benzothiazole cyanine dyes, the cyanine dyes of the present invention have high biological penetrance and are more suitable for cell dyes and imaging.

[0015] Compound

[0016] In a first aspect, the present application provides a compound having a structure as shown in general formula I or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof,

[0017] in,

[0018] R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 A linear or branched alkylene group -M, wherein M is selected from sulfonic acid group, phenyl group, carboxyl group, mercapto group, and amino group;

[0019] R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 alkyl;

[0020] Y does not exist or is a counter anion;

[0021] And limit:

[0022] When R3 is hydrogen, R1 and R2 are not both methyl and R1 and R2 are not both benzyl.

[0023] In the compounds of the present invention, R1 and R2 may be the same or different. In some embodiments, R1 and R2 are independently selected from C 1-6 Straight chain alkyl, C 1-6 The linear alkylene group -M, M is selected from sulfonic acid group, phenyl group, carboxyl group, mercapto group, and amino group.

[0024] In some embodiments, at least one of R1 and R2 is C 1-18 Straight-chain or branched alkylene-sulfonic acid group.

[0025] In some embodiments, R1 and R2 are different and independently selected from C 1-6 Straight chain alkyl, benzyl, C 1-6 Straight chain alkylene-carboxyl, C1-6 Straight chain alkylene-sulfonic acid group, C 1-6 Straight chain alkylene-mercapto, C 1-6 Straight chain alkylene-amino.

[0026] In some embodiments, R1 and R2 are the same and are selected from C 1-6 Straight chain alkyl, C 1-6 Straight chain alkylene-sulfonic acid group, C 1-6 Straight chain alkylene-carboxyl.

[0027] In some embodiments, R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 alkyl.

[0028] In some embodiments, Y in Formula I is a counter anion, in which case Y can be selected from a halogen ion (e.g., F - 、Cl - Br - , I - )、ClO4 - PF6 - CF3SO3 - 、BF4 - , acetate, methanesulfonate or p-toluenesulfonate.

[0029] In some embodiments, Y in Formula I is absent, in which case the compound may be an inner salt. "Inner salts" are also referred to as "zwitterions" in the art. In some embodiments, the compounds of the present invention may contain both an acidic group (e.g., a sulfonic acid group or a carboxyl group) and a basic group (e.g., an amino group or a thiazole ring) within the molecule, and the acidic and basic groups neutralize each other to form an inner salt.

[0030] It should be understood that when a compound molecule contains multiple acidic groups and / or multiple basic groups, each acidic group and / or each basic group may serve as a salt-forming group. Salts formed by various salt-forming methods are all within the scope of the present invention.

[0031] In some embodiments, at least one of R1 and R2 is selected from C 1-18 Straight chain or branched chain alkylene-sulfonic acid group, C 1-18 Straight-chain or branched alkylene-carboxyl.

[0032] The compounds of the present invention may have any of the following structures:

[0033] In some embodiments, the compound of the present invention is a compound represented by the above structural formula 5, 6 or 9.

[0034] Conjugates and compositions for staining biological samples

[0035] The compounds of the present invention and their hydrates, solvates, stereoisomers, tautomers or crystal forms can be used directly for staining biological samples, or can be used in the form of derivatives, including but not limited to conjugates.

[0036] As used herein, "conjugate" refers to a compound of the present invention, its hydrate, solvate, stereoisomer, tautomer or crystal form connected to other molecules by a covalent bond. The molecule that can be conjugated to the compound of the present invention, its hydrate, solvate, stereoisomer, tautomer or crystal form can be a molecule that specifically binds to a cell or a cell component, including but not limited to antibodies, antigens, receptors, ligands, enzymes, substrates, coenzymes, etc. Typically, the test sample is incubated with the fluorescent conjugate for a period of time so that the fluorescent conjugate specifically binds to certain cells or cell components in the test sample. The binding of the fluorescent conjugate to cells or cell components can also be referred to as staining. The staining step can be performed multiple times in sequence, or multiple stainings can be performed simultaneously with multiple conjugates. After staining is completed, the sample is analyzed in an analytical instrument comprising an excitation light source and a measuring device, wherein the excitation light source excites the fluorescent dye of the present invention in the conjugate, and the measuring device measures the emitted light generated by the excited fluorescent dye.

[0037] The application also provides a composition for staining a biological sample, wherein the composition comprises a compound of the present invention, a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, or a conjugate of the present invention. In some embodiments, the biological sample is a nucleic acid. In some embodiments, the biological sample is a deoxyribonucleic acid.

[0038] use

[0039] The present application also provides the use of a compound of the present invention, a hydrate, solvate, stereoisomer, tautomer, or crystal form thereof, or a conjugate of the present invention, or a composition of the present invention for staining a biological sample. In some embodiments, the biological sample is a nucleic acid. In some embodiments, the biological sample is a deoxyribonucleic acid.

[0040] The present application also provides the use of a compound of the present invention or a hydrate, solvate, stereoisomer, tautomer, or crystal form thereof, or a conjugate of the present invention, or a composition of the present invention in identifying parasites in a blood sample to be tested using flow cytometry. In some embodiments, the parasite is selected from the group consisting of Ascaris lumbricoides, hookworms, tapeworms, Trichomonas vaginalis, liver flukes, Paragonimus westermannii, Toxoplasma gondii, Cysticercus pombe, Trichinella spiralis, Amoeba, Leishmania donovani, Plasmodium, Schistosoma japonicum, Filarial worms, Hydatid cysts, Scabies mites, Trichomonas follicularis, lice, and fleas.

[0041] The present application also provides use of the compound of the present invention or its hydrate, solvate, stereoisomer, tautomer or crystal form, or the conjugate of the present invention, or the composition of the present invention in identifying microorganisms in a blood sample or a body fluid sample to be tested using flow cytometry.

[0042] In some embodiments, the microorganism is selected from:

[0043] Bacteria (e.g., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Bordetella pertussis, Corynebacterium diphtheriae, Neisseria meningitidis, Mycobacterium tuberculosis, Clostridium tetani, Mycobacterium leprae, Group A hemolytic Streptococcus, Brucella, Clostridium cholerae, Salmonella typhi, Bacillus anthracis, Neisseria gonorrhoeae, Vibrio cholerae, Pseudomonas Klebsiella, and Salmonella paratyphi A, B, or C),

[0044] Viruses (e.g. influenza virus, mumps virus, rubella virus, Japanese encephalitis virus, dengue virus, hemorrhagic fever virus, rabies virus, human papillomavirus, poliovirus, measles virus, varicella-zoster virus, hepatitis virus, enterovirus 70, coxsackievirus A24 variant, human immunodeficiency virus, poxvirus (e.g. monkeypox virus)),

[0045] Fungi (e.g., Candida albicans, Trichophyton rubrum, Epidermophyton floccosum),

[0046] Mycoplasma (e.g., Mycoplasma pneumoniae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium),

[0047] Chlamydia (e.g., Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia tumefaciens),

[0048] Rickettsiae (e.g., Rickettsia prowazekii, Rickettsia moorei, Rickettsia rickettsii, Rickettsia tsutsugamushi),

[0049] Actinomycetes (e.g. Actinomycetes israelii),

[0050] Spirochetes (e.g., Leptospira, Treponema pallidum).

[0051] In some embodiments, blood or body fluid refers to blood or body fluid from mammals, especially humans. Body fluids can be divided into urine, sweat, cerebrospinal fluid, serous cavity fluid, synovial fluid, etc., depending on the location. A normal human body contains small amounts of all of the above body fluids. Effusions in serous cavities include pleural effusions, ascites, and pericardial effusions, while effusions in joint cavities are synovial fluids.

[0052] General synthetic method

[0053] The compounds of the present invention can be synthesized by common methods in the art. For example, the benzothiazole compounds of the present invention can be synthesized by the following method: first, unsubstituted or substituted methylbenzothiazole is reacted with R2X (X is F, Cl, Br, or I) under heating and reflux to obtain an intermediate I in the form of a quaternary ammonium salt. Subsequently, a linker molecule 4-hydroxyisophthalaldehyde is reacted with the intermediate I under heating and reflux (the molar ratio of the intermediate I to 4-hydroxyisophthalaldehyde is greater than or equal to 2), so that the intermediate I and the linker molecule condense to obtain the benzothiazole compound of the present invention.

[0054] When R2 is C 1-18 In the case of a linear or branched alkylene-sulfonic acid group, intermediate I can also be synthesized by the following method: starting from a raw material such as unsubstituted or substituted methylbenzothiazole, intermediate I is obtained by reacting it with a suitable sultone through a ring-opening reaction.

[0055] The above method is suitable for the case where R1 and R2 are the same.

[0056] When R1 and R2 are different, the benzothiazole compounds of the present invention can be synthesized by the following exemplary method: heating and reflux reaction of methylbenzothiazole substituted with R1 or R2 and a linker molecule 4-hydroxyisophthalaldehyde (the molar ratio of 4-hydroxyisophthalaldehyde to methylbenzothiazole substituted with R1 or R2 is approximately 2) to obtain a formyl group-containing intermediate I'; and condensing the obtained intermediate I' with methylbenzothiazole substituted with R2 or R1 to obtain the benzothiazole compounds of the present invention.

[0057] In the above method, each intermediate or product can be recovered by separation and purification techniques known in the art to achieve the required purity.

[0058] Various raw materials used in the above methods are commercially available, or can be prepared from raw materials known in the art by methods known to those skilled in the art or methods disclosed in the prior art.

[0059] Definition of terms

[0060] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the experimental procedures used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0061] As used herein, the term “C 1-18 The term "straight-chain or branched-chain alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched-chain alkane containing 1 to 18 carbon atoms, and specific examples include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, tert-butyl, isobutyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, and n-octadecyl.

[0062] As used herein, “C 1-6 "Alkyl" refers to a group obtained by removing a hydrogen atom from a straight or branched chain alkane containing 1 to 6 carbon atoms, and specific examples include but are not limited to methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, tert-butyl, isobutyl, etc.

[0063] As used herein, “C 1-6 The term "straight-chain alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain alkane containing 1 to 6 carbon atoms, and specific examples thereof include but are not limited to methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl.

[0064] C 1-18 A straight chain or branched chain alkylene group refers to a group obtained by removing two hydrogen atoms from a straight chain or branched chain alkane containing 1 to 18 carbon atoms, and specific examples thereof include but are not limited to methylene, ethylene, propylene, butylene, etc. The term "C 1-6 The term "straight-chain alkylene" refers to a group obtained by removing two hydrogen atoms from a straight-chain alkane containing 1 to 6 carbon atoms.

[0065] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0066] As used herein, the term "halogenated" refers to a group or compound in which hydrogen is replaced by one or more halogen atoms, including full halogenation and partial halogenation.

[0067] As used herein, the term “C 1-6 "Alkoxy" refers to a group formed in the form of C1-6 alkyl-O-.

[0068] As used herein, the term "solvate" (or "solvate") refers to a substance formed by the association of a compound with organic solvent molecules (eg, methanol, ethanol, propanol, acetonitrile, etc.).

[0069] As used herein, the term "hydrate" refers to a substance formed by the association of a compound with water molecules.

[0070] As used herein, the term "crystalline form" refers to the crystal structure of a substance. During crystallization, various factors may alter the intramolecular or intermolecular bonding patterns, resulting in different arrangements of molecules or atoms within the crystal lattice space, forming different crystal structures. The compounds of the present invention may exist in a single crystal structure or in multiple crystal structures, i.e., they may exhibit "polymorphism." The compounds of the present invention may exist in different crystalline forms.

[0071] As used herein, the term "stereoisomer" includes conformational isomers and configurational isomers, wherein the configurational isomers primarily include cis-trans isomers and optical isomers. The compounds of the present invention may exist in the form of stereoisomers, and therefore encompass all possible stereoisomeric forms, and any combination or any mixture thereof. For example, a single enantiomer, a single diastereomer, or a mixture thereof. When a compound of the present invention contains an olefin double bond, unless otherwise specified, it includes cis-isomers and trans-isomers, and any combination thereof.

[0072] Compound of the present invention can exist with tautomer form, and it has the connection point of different hydrogen by one or more double bond displacements.For example, ketone and its enol form are keto-enol tautomers.Should be understood that the present invention comprises the keto-enol tautomers of all compounds.Each tautomer and mixture thereof are all included in the scope of the present invention.

[0073] Advantageous Effects of the Invention

[0074] Compared with the prior art, the fluorescent dye of the present invention has one or more of the following beneficial effects:

[0075] 1. Compared with existing dyes, the thermal stability of the dyes of the present invention is significantly improved;

[0076] 2. Dyes for cell staining require high cell penetration. Compared with existing dyes, the dyes of the present invention have high biological penetration and are more suitable for cell staining and imaging.

[0077] 3. The dye of the present invention can specifically bind to DNA, which is beneficial for the specific recognition and accurate measurement of DNA;

[0078] 4. The dye of the present invention has good permeability to living cells and can enter cells to dye nucleic acids without destroying the cell membrane. It has low toxicity and low carcinogenicity.

[0079] 5. The excitation light of the dye of the present invention is blue-green light with a shorter wavelength, which can identify tiny particles and improve the detection ability of small particles;

[0080] 6. The dye of the present invention can use ordinary green semiconductor lasers as light sources, which greatly reduces the cost of use;

[0081] 7. The dye of the present invention has a simple structure, the raw materials for its preparation are readily available, the synthesis yield is high, and it is easy to realize industrialization.

[0082] To solve the above problems, another object of the present invention is to provide a sample analysis method and a sample analyzer, which can accurately, sensitively, cheaply and quickly identify microorganisms in blood or body fluids based on flow cytometry and using fluorescent labeling technology.

[0083] To this end, a second aspect of the present invention provides a sample analysis method, comprising the following steps:

[0084] Treating the same biological sample to be tested with a dye reagent containing a first dye and a hemolytic agent for dissolving red blood cells to obtain a sample solution to be tested, wherein the biological sample to be tested is a blood sample to be tested or a body fluid sample to be tested, and the first dye can stain microorganisms;

[0085] Allowing particles in the sample liquid to pass through an optical detection zone one by one and irradiating the particles passing through the optical detection zone with light, so as to obtain scattered light information and fluorescence information generated by the particles in the sample liquid after being irradiated with light, wherein the fluorescence information includes first fluorescence information from the first dye; and

[0086] The microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

[0087] In some embodiments, identifying the microorganisms in the sample liquid based on the scattered light information and the first fluorescence information includes:

[0088] generating a first scattergram based on the scattered light information and the first fluorescence information; and

[0089] Microorganisms in the sample liquid to be tested are identified based on the first scattergram.

[0090] In some embodiments, the scattered light information includes forward scattered light information; and

[0091] Generating a first scattergram based on the scattered light information and the first fluorescence information includes: generating the first scattergram based on the forward scattered light information and the first fluorescence information.

[0092] In some embodiments, identifying the microorganisms in the sample liquid based on the scattered light information and the first fluorescence information includes:

[0093] Acquire a microorganism characteristic region and a white blood cell region from the first scattergram, wherein the intensity of the first fluorescence information of the microorganism characteristic region is greater than the intensity of the first fluorescence information of the white blood cell region; and

[0094] The microorganisms in the sample liquid to be tested are identified based on the microorganism characteristic region.

[0095] In some embodiments, identifying the microorganisms in the sample liquid based on the scattered light information and the first fluorescence information includes:

[0096] The number of microorganisms in the sample liquid to be tested is obtained based on the scattered light information and the first fluorescence information.

[0097] In some embodiments, the biological sample to be tested is a blood sample to be tested, and the sample analysis method further comprises:

[0098] The parasites, especially Plasmodium, in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

[0099] In some embodiments, the biological sample to be tested is a blood sample to be tested, the dye reagent further includes a second dye different from the first dye, and the fluorescence information further includes second fluorescence information from the second dye, wherein the second dye can stain white blood cells in the blood; and

[0100] The sample analysis method further includes: obtaining a white blood cell classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

[0101] In some embodiments, the scattered light information includes side scattered light information and forward scattered light information;

[0102] Identifying the microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information includes: identifying the microorganisms in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information; and

[0103] Acquiring the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information includes: acquiring the leukocyte classification result of the sample liquid to be tested based on the side scattered light information and the second fluorescence information.

[0104] In some embodiments, the biological sample to be tested is a blood sample to be tested, the dye reagent further includes a second dye different from the first dye, and the fluorescence information further includes second fluorescence information from the second dye, wherein the second dye can stain white blood cells and nucleated red blood cells in the blood; and

[0105] The sample analysis method further includes: obtaining at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

[0106] In some embodiments, the scattered light information includes forward scattered light information;

[0107] Identifying the microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information includes: identifying the microorganisms in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information; and

[0108] Obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information includes: obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the forward scattered light information and the second fluorescence information.

[0109] In some embodiments, illuminating the particles flowing through the optical detection zone with light comprises illuminating the particles flowing through the optical detection zone with light of a single wavelength.

[0110] In some embodiments, the first dye is a dye that can specifically bind to deoxyribonucleic acid.

[0111] In some embodiments, the first dye comprises a compound having the structure of the above-mentioned general formula I, wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino, R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Y is absent or is a counter anion.

[0112] For more details and advantages of this compound, please refer to the above description and will not be repeated here.

[0113] A third aspect of the present invention provides a sample analyzer, comprising:

[0114] A sampling device for quantitatively drawing a biological sample to be tested, wherein the biological sample to be tested is a blood sample to be tested or a body fluid sample to be tested;

[0115] A sample preparation device having a reaction pool and a reagent supply unit, wherein the reaction pool is used to receive the biological sample to be tested sucked by the sampling device, and receive a dye reagent containing a first dye and a hemolytic agent for dissolving red blood cells provided by the reagent supply unit. The biological sample to be tested sucked by the sampling device and the dye reagent and hemolytic agent provided by the reagent supply unit are mixed in the reaction pool to prepare a sample liquid to be tested, wherein the first dye can stain microorganisms;

[0116] an optical detection device, comprising a light source, a flow cell, a scattered light detector, and a fluorescence detector, wherein the light source is configured to emit a light beam to illuminate the flow cell, the flow cell being in communication with the reaction pool and allowing particles in the sample liquid to pass through the flow cell one by one, the scattered light detector being configured to detect scattered light information generated by particles passing through the flow cell after being illuminated by light, and the fluorescence detector being configured to detect fluorescence information generated by particles passing through the flow cell after being illuminated by light, the fluorescence information including first fluorescence information from the first dye; and

[0117] The processor is configured to obtain the scattered light information and the fluorescence information from the optical detection device, and identify microorganisms in the sample liquid based on the scattered light information and the first fluorescence information.

[0118] In some embodiments, the processor is further configured to perform the following steps when identifying microorganisms in the sample liquid based on the scattered light information and the first fluorescence information:

[0119] generating a first scattergram based on the scattered light information and the first fluorescence information; and

[0120] Microorganisms in the sample liquid to be tested are identified based on the first scattergram.

[0121] In some embodiments, the scattered light information includes forward scattered light information; and

[0122] The processor is further configured to generate the first scattergram based on the forward scattered light information and the first fluorescence information.

[0123] In some embodiments, the processor is further configured to:

[0124] Acquire a microorganism characteristic region and a white blood cell region from the first scattergram, wherein the intensity of the first fluorescence information of the microorganism characteristic region is greater than the intensity of the first fluorescence information of the white blood cell region; and

[0125] The microorganisms in the sample liquid to be tested are identified based on the microorganism characteristic region.

[0126] In some embodiments, the processor is further configured to perform the following steps when identifying microorganisms in the sample liquid based on the scattered light information and the first fluorescence information:

[0127] The number of microorganisms in the sample liquid to be tested is obtained based on the scattered light information and the first fluorescence information.

[0128] In some embodiments, the biological sample to be tested is a blood sample to be tested, and the processor is further configured to:

[0129] The parasites, especially Plasmodium, in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

[0130] In some embodiments, the biological sample to be tested is a blood sample to be tested, the dye reagent further includes a second dye different from the first dye, the second dye can stain white blood cells in the blood, and the fluorescence information further includes second fluorescence information from the second dye; and

[0131] The processor is further configured to obtain a white blood cell classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

[0132] In some embodiments, the scattered light information includes side scattered light information and forward scattered light information; and

[0133] The processor is further configured to: identify microorganisms in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information, and obtain a leukocyte classification result of the sample liquid to be tested based on the side scattered light information and the second fluorescence information.

[0134] In some embodiments, the biological sample to be tested is a blood sample to be tested, the dye reagent further includes a second dye different from the first dye, the second dye can stain white blood cells and nucleated red blood cells in the blood, and the fluorescence information further includes second fluorescence information from the second dye; and

[0135] The processor is further configured to obtain at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

[0136] In some embodiments, the scattered light information includes forward scattered light information; and

[0137] The processor is further configured to: identify microorganisms in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information, and obtain at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample liquid to be tested based on the forward scattered light information and the second fluorescence information.

[0138] In some embodiments, the light source is configured to illuminate the flow chamber with light of a single wavelength.

[0139] In some embodiments, the first dye is a dye that can specifically bind to deoxyribonucleic acid.

[0140] In some embodiments, the first dye comprises a compound having the structure of the above-mentioned general formula I, wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino, R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Y is absent or is a counter anion.

[0141] For more details and advantages of this compound, please refer to the above description and will not be repeated here.

[0142] In the sample analysis method of the second aspect and the sample analyzer of the third aspect of the present invention, a blood sample or body fluid sample is first treated with a dye capable of staining microorganisms and a hemolytic agent for dissolving red blood cells to obtain a test sample liquid. Flow cytometry is then used to obtain scattered light information and fluorescence information from the test sample liquid. Based on the scattered light information and fluorescence information, the presence of microorganisms in the test sample liquid can be identified. This enables accurate, sensitive, cost-effective, and rapid detection of microorganisms in blood or body fluids.

[0143] In order to solve the above problems, another task of the present invention is to provide a blood analysis method and a blood analyzer, which can simply, sensitively, quickly and accurately detect parasites in the blood during a low-cost routine blood test based on flow cytometry and using fluorescent labeling technology.

[0144] To this end, the fourth aspect of the present invention provides a blood analysis method comprising the following steps:

[0145] A dye reagent comprising a first dye and a hemolytic agent for dissolving red blood cells are used to treat the same blood sample to obtain a sample solution to be tested, wherein the first dye can stain parasites in the blood, and the first dye comprises a compound having the structure of the above-mentioned general formula I, wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino, R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Y is absent or is a counter anion;

[0146] Allowing particles in the sample liquid to pass through an optical detection zone one by one and irradiating the particles passing through the optical detection zone with light, so as to obtain scattered light information and fluorescence information generated by the particles in the sample liquid after being irradiated with light, wherein the fluorescence information includes first fluorescence information from the first dye; and

[0147] The parasites in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

[0148] In some embodiments, identifying parasites in the sample liquid based on the scattered light information and the first fluorescence information includes:

[0149] generating a first scattergram based on the scattered light information and the first fluorescence information; and

[0150] The parasites in the sample liquid to be tested are identified based on the first scatter plot.

[0151] In some embodiments, the scattered light information includes forward scattered light information; and

[0152] Generating a first scattergram based on the scattered light information and the first fluorescence information includes: generating the first scattergram based on the forward scattered light information and the first fluorescence information.

[0153] In some embodiments, identifying parasites in the sample liquid based on the scattered light information and the first fluorescence information includes:

[0154] Acquire a parasite characteristic region and a leukocyte region from the first scattergram, wherein the intensity of the first fluorescence information of the parasite characteristic region is greater than the intensity of the first fluorescence information of the leukocyte region; and

[0155] The parasites in the sample liquid to be tested are identified based on the parasite characteristic region.

[0156] In some embodiments, identifying parasites in the sample liquid based on the scattered light information and the first fluorescence information includes:

[0157] The number of parasites in the sample liquid to be tested is obtained based on the scattered light information and the first fluorescence information.

[0158] In some embodiments, the blood analysis method further comprises:

[0159] The microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

[0160] In some embodiments, the compound according to the first aspect of the present invention can be used as the compound contained in the first dye. For more details and advantages of the compound, please refer to the above description and will not be repeated here.

[0161] In some embodiments, the dye reagent further comprises a second dye different from the first dye, the second dye being capable of staining white blood cells and nucleated red blood cells in the blood, and the fluorescence information further comprises second fluorescence information from the second dye; and

[0162] The blood analysis method further includes: obtaining at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

[0163] In some embodiments, the scattered light information includes forward scattered light information;

[0164] Identifying the parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information includes: identifying the parasites in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information; and

[0165] Obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information includes: obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the forward scattered light information and the second fluorescence information.

[0166] In some embodiments, the dye reagent further comprises a second dye different from the first dye, the second dye being capable of staining white blood cells in the blood, and the fluorescence information further comprises second fluorescence information from the second dye; and

[0167] The blood analysis method further includes: obtaining a white blood cell classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

[0168] In some embodiments, the scattered light information includes side scattered light information and forward scattered light information;

[0169] Identifying the parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information includes: identifying the parasites in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information; and

[0170] Acquiring the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information includes: acquiring the leukocyte classification result of the sample liquid to be tested based on the side scattered light information and the second fluorescence information.

[0171] In some embodiments, illuminating the particles flowing through the optical detection zone with light comprises illuminating the particles flowing through the optical detection zone with light of a single wavelength.

[0172] A fifth aspect of the present invention provides a blood analyzer, comprising:

[0173] A sampling device, used for quantitatively drawing a blood sample to be tested;

[0174] The sample preparation device comprises a reaction pool and a reagent supply portion, wherein the reaction pool is used to receive the blood sample to be tested drawn by the sampling device, and receive a dye reagent containing a first dye and a hemolytic agent for dissolving red blood cells provided by the reagent supply portion. The blood sample to be tested drawn by the sampling device and the dye reagent and hemolytic agent provided by the reagent supply portion are mixed in the reaction pool to prepare a sample solution to be tested, wherein the first dye comprises a compound having the structure of the above-mentioned general formula I, wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino, R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Y is absent or is a counter anion;

[0175] an optical detection device, comprising a light source, a flow cell, a scattered light detector, and a fluorescence detector, wherein the light source is configured to emit a light beam to illuminate the flow cell, the flow cell being in communication with the reaction pool and allowing particles in the sample liquid to pass through the flow cell one by one, the scattered light detector being configured to detect scattered light information generated by particles passing through the flow cell after being illuminated by light, and the fluorescence detector being configured to detect fluorescence information generated by particles passing through the flow cell after being illuminated by light, the fluorescence information including first fluorescence information from the first dye; and

[0176] The processor is configured to obtain the scattered light information and the fluorescence information from the optical detection device, and identify parasites in the sample liquid based on the scattered light information and the first fluorescence information.

[0177] In some embodiments, the processor is further configured to perform the following steps when identifying parasites in the sample fluid based on the scattered light information and the first fluorescence information:

[0178] generating a first scattergram based on the scattered light information and the first fluorescence information; and

[0179] The parasites in the sample liquid to be tested are identified based on the first scatter plot.

[0180] In some embodiments, the scattered light information includes forward scattered light information; and

[0181] The processor is further configured to generate the first scattergram based on the forward scattered light information and the first fluorescence information.

[0182] In some embodiments, the processor is further configured to:

[0183] Acquire a parasite characteristic region and a leukocyte region from the first scattergram, wherein the intensity of the first fluorescence information of the parasite characteristic region is greater than the intensity of the first fluorescence information of the leukocyte region; and

[0184] The parasites in the sample liquid to be tested are identified based on the parasite characteristic region.

[0185] In some embodiments, the processor is further configured to perform the following steps when identifying parasites in the sample fluid based on the scattered light information and the first fluorescence information:

[0186] The number of parasites in the sample liquid to be tested is obtained based on the scattered light information and the first fluorescence information.

[0187] In some embodiments, the processor is further configured to:

[0188] The microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

[0189] In some embodiments, the compound according to the first aspect of the present invention can be used as the compound contained in the first dye. For more details and advantages of the compound, please refer to the above description and will not be repeated here.

[0190] In some embodiments, the dye reagent further comprises a second dye different from the first dye, the second dye being capable of staining white blood cells and nucleated red blood cells in the blood, and the fluorescence information further comprises second fluorescence information from the second dye; and

[0191] The processor is further configured to obtain at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

[0192] In some embodiments, the scattered light information includes forward scattered light information; and

[0193] The processor is further configured to: identify parasites in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information, and obtain at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample liquid to be tested based on the forward scattered light information and the second fluorescence information.

[0194] In some embodiments, the dye reagent further comprises a second dye different from the first dye, the second dye being capable of staining white blood cells in the blood, and the fluorescence information further comprises second fluorescence information from the second dye; and

[0195] The processor is further configured to obtain a white blood cell classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

[0196] In some embodiments, the scattered light information includes side scattered light information and forward scattered light information; and

[0197] The processor is further configured to: identify parasites in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information, and obtain a white blood cell classification result of the sample liquid to be tested based on the side scattered light information and the second fluorescence information.

[0198] In some embodiments, the light source is configured to illuminate the flow chamber with light of a single wavelength.

[0199] In the sample analysis method and sample analyzer of the fourth aspect of the present invention and the fifth aspect, a blood sample is first treated with a cyanine dye capable of staining parasites, particularly a benzothiazole-based cyanine dye, and a hemolytic agent for dissolving red blood cells to obtain a sample solution. Flow cytometry is then used to obtain scattered light and fluorescence information from the sample solution. The presence of parasites in the sample solution can be determined based on the scattered light and fluorescence information. This allows for accurate, sensitive, cost-effective, and rapid detection of parasites in blood.

[0200] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0201] FIG1 is a schematic flow chart of an embodiment of a sample analysis method for identifying microorganisms according to the present invention.

[0202] FIG. 2 is a first scattergram when the biological sample to be tested is a blood sample according to an embodiment of the present invention.

[0203] FIG3 is a first scattergram when the biological sample to be tested is a body fluid sample according to an embodiment of the present invention.

[0204] FIG4 is a schematic flow chart of another embodiment of a sample analysis method for identifying microorganisms according to the present invention.

[0205] FIG5 is a scatter plot of microorganism identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to the present invention.

[0206] FIG6 is a schematic flow chart of yet another embodiment of a sample analysis method for identifying microorganisms according to the present invention.

[0207] FIG. 7 is a scatter plot of microorganism identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to the present invention.

[0208] FIG8 is a schematic diagram of emission spectra of two dyes according to an embodiment of the present invention.

[0209] FIG9 is a schematic diagram of the emission spectrum and excitation spectrum of a large Stokes shift dye according to an embodiment of the present invention.

[0210] FIG10 is a schematic flow chart of an embodiment of a blood analysis method for identifying parasites according to the present invention;

[0211] FIG11 is a first scatter plot for identifying parasites according to an embodiment of the present invention;

[0212] FIG12 is a schematic flow chart of another embodiment of a blood analysis method for identifying parasites according to the present invention;

[0213] FIG13 is a scatter plot showing the parasite identification results and the nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to the present invention;

[0214] FIG14 is a schematic flow chart of yet another embodiment of a blood analysis method for identifying parasites according to the present invention;

[0215] FIG15 is a scatter plot showing parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to the present invention;

[0216] FIG. 16 is a schematic structural diagram of a sample analyzer according to an embodiment of the present invention.

[0217] 17 to 19 are schematic structural diagrams of different embodiments of the optical detection device according to the present invention.

[0218] FIG20 shows the change in fluorescence spectrum of the dye in Example 7 as the DNA concentration increases.

[0219] FIG21 shows the change in fluorescence spectrum of the dye in Example 7 as the RNA concentration increases.

[0220] FIG22 is a graph showing the linear relationship between fluorescence intensity and calf thymus DNA and RNA concentrations in Example 7.

[0221] FIG23 shows the results of staining of HepG2 living cells by Compound II observed under a laser microscope in Example 8.

[0222] FIG24 shows the results of staining of HepG2 living cells by Compound III observed under a laser microscope in Example 9.

[0223] FIG. 25 shows the evaluation results of dye stability in Example 10.

[0224] FIG26 shows the evaluation results of the cell penetration ability of the dye in Example 11.

[0225] FIG27 is a scatter plot of microorganism identification results and white blood cell classification results obtained simultaneously in one test of the same blood sample according to Example 12 of the present invention.

[0226] FIG. 28 is a scatter plot obtained by testing body fluid samples according to Example 13 of the present invention.

[0227] FIG29 is a scatter plot of microorganism identification results and white blood cell classification results obtained simultaneously in a single test of the same blood sample according to Example 14 of the present invention.

[0228] FIG30 is a scatter plot obtained by testing body fluid samples according to Example 15 of the present invention.

[0229] FIG31 is a scatter plot of microorganism identification results and white blood cell classification results obtained simultaneously in one test of the same blood sample according to Example 16 of the present invention.

[0230] FIG32 is a scatter plot obtained by testing body fluid samples according to Example 17 of the present invention.

[0231] FIG33 is a scatter plot of microorganism identification results and nucleated red blood cell identification results obtained simultaneously in a single test of the same blood sample according to Example 18 of the present invention.

[0232] FIG34 is a scatter plot obtained by testing body fluid samples according to Example 19 of the present invention.

[0233] FIG35 is a scatter plot of microorganism identification results and nucleated red blood cell identification results obtained simultaneously in a single test of the same blood sample according to Example 20 of the present invention.

[0234] FIG36 is a scatter plot obtained by testing body fluid samples according to Example 21 of the present invention.

[0235] FIG37 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in a single test of the same blood sample according to Example 22 of the present invention.

[0236] FIG38 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in a single test of the same blood sample according to Example 23 of the present invention.

[0237] FIG39 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in a single test of the same blood sample according to Example 24 of the present invention.

[0238] FIG40 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in a single test of the same blood sample according to Example 25 of the present invention.

[0239] FIG41 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in a single test of the same blood sample according to Example 26 of the present invention.

[0240] FIG42 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in a single test of the same blood sample according to Example 27 of the present invention. DETAILED DESCRIPTION

[0241] The following will clearly and completely describe the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0242] It should be noted that the terms "first\second\third" involved in the embodiments of the present invention are only used to distinguish similar objects and do not represent a specific order for the objects. It can be understood that "first\second\third" can be interchanged with a specific order or sequence when permitted.

[0243] As an essential instrument for routine blood analysis in modern clinical hematology, the blood cell analyzer (also known as blood analyzer or blood cell analyzer) based on flow cytometry has the advantages of fast, accurate and extremely simple operation for blood cell detection. It is an indispensable fully automated blood cell rapid detection instrument in modern clinical testing.

[0244] Based on this, the present invention proposes a technical solution for rapidly detecting pathogenic microorganisms in blood or body fluids based on flow cytometry and fluorescent labeling technology.

[0245] In the embodiments of the present invention, microorganisms refer to tiny organisms that are difficult to see with the naked eye and can only be observed with the aid of an optical microscope or an electron microscope. Microorganisms include bacteria, viruses, fungi, and a few algae.

[0246] As shown in FIG1 , an embodiment of the present invention first provides a sample analysis method 100 for rapidly detecting microorganisms in blood or body fluids based on flow cytometry and fluorescent labeling technology. The sample analysis method 100 includes the following steps S110 , S120 , and S130 .

[0247] In step S110, a dye reagent containing a first dye and a hemolytic agent for dissolving red blood cells are used to treat the same biological sample to obtain a sample solution. The biological sample to be tested is a blood sample or a body fluid sample. The first dye is capable of staining microorganisms. That is, when microorganisms are present in the blood sample or body fluid sample, treating the blood sample or body fluid sample with the first dye stains the microorganisms.

[0248] For example, in step S110, a hemolytic agent and a dye reagent may be added sequentially to the same blood sample or body fluid sample to be tested to obtain a test sample solution, and then the test sample solution may be incubated so that the dye reagent can fully stain the test substance in the test sample solution. For another example, in step S110, the dye reagent may be pre-mixed with the hemolytic agent to obtain a mixed reagent, and then the mixed reagent and the biological sample to be tested may be mixed at a volume ratio of 250:1 to 1000:1. After the mixture is uniformly mixed, the mixed test sample solution may be incubated at a temperature of 25°C to 50°C for 10 seconds to 1 minute, preferably for 20 seconds to 40 seconds.

[0249] It can be understood that in the embodiment of the present invention, the hemolytic agent is used to dissolve the red blood cells in the blood or body fluids and break the red blood cells into fragments, but can keep the morphology of the white blood cells basically unchanged.

[0250] In some embodiments, the hemolytic agent may comprise any one or a combination of a cationic surfactant, a nonionic surfactant, an anionic surfactant, an amphiphilic surfactant, or a buffer pair. Cationic surfactants are, for example, selected from at least one or a combination of dodecyltrimethylammonium chloride, octyltrimethylammonium bromide, and tetradecyltrimethylammonium chloride. Nonionic surfactants are, for example, selected from at least one or a combination of long-chain fatty alcohol polyoxyethylene, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ether, and fatty amine polyoxyethylene ether. Buffer pairs are, for example, selected from at least one or a combination of phosphates, citrates, and Tris-HCl. Anionic surfactants are, for example, selected from at least one or a combination of dodecylbenzenesulfonic acid, sodium fatty alcohol sulfate, sodium ethoxylated fatty acid methyl ester sulfonate, sodium secondary alkyl sulfonate, and alcohol ether carboxylates.

[0251] In other embodiments, the hemolytic agent may include at least one of an alkyl glycoside, a triterpenoid saponin, and a steroidal saponin.

[0252] In step S120, particles in the sample solution are passed through an optical detection zone one by one, and light is applied to the particles passing through the optical detection zone to obtain scattered light information and fluorescence information generated by the particles in the sample solution after being exposed to light. The fluorescence information includes at least first fluorescence information from the first dye, that is, the fluorescence signal generated by the particles in the sample solution after binding to the first dye under light excitation.

[0253] That is, in step S120, scattered light information and fluorescence information of the sample fluid to be tested are obtained based on the principles of flow cytometry. When light, such as a laser beam, illuminates particles flowing through the optical detection zone, the particle's inherent characteristics (such as volume, staining level, size and content of cellular contents, and nuclear density) can block or redirect the laser beam, thereby generating scattered light at various angles corresponding to the particle's characteristics. This scattered light is received by a signal detector, and optical information related to the particle's structure and composition, i.e., the scattered light information and fluorescence information of the present invention, can be obtained. Here, the scattered light information, for example, includes at least one of forward scattered light information and side scattered light information. Forward scattered light reflects the number and volume of particles, side scattered light (SS) reflects the complexity of the cell's internal structure (such as intracellular granules or nuclei), and fluorescence (FL) reflects the content of nucleic acid in the cell. Using this optical information, various types of particles in the sample fluid to be tested can be identified.

[0254] In an embodiment of the present invention, the scattered light information includes scattered light signal intensity, and the fluorescence information includes fluorescence signal intensity.

[0255] In step S130 , microorganisms in the sample liquid are identified based on the scattered light information and the first fluorescence information.

[0256] In some embodiments, step S130 , identifying microorganisms in the sample liquid based on the scattered light information and the first fluorescence information, may include: generating a first scattergram based on the scattered light information and the first fluorescence information; and identifying microorganisms in the sample liquid based on the first scattergram.

[0257] In an embodiment of the present invention, the scatter plot can be a two-dimensional scatter plot or a three-dimensional scatter plot, on which two-dimensional or three-dimensional characteristic information of multiple particles is distributed, wherein the X-axis, Y-axis, and Z-axis of the scatter plot each represent a characteristic of each particle. For example, in a scatter plot, the X-axis represents the forward scattered light signal intensity, the Y-axis represents the fluorescence signal intensity, and the Z-axis represents the side scattered light signal intensity. It should be noted that the scatter plot herein is not limited to a graphical form, and can also be in a data form, such as a digital form of a table or list with the same or similar resolution as the scatter plot, or presented in any other suitable manner known in the art.

[0258] Furthermore, the scattered light information may include forward scattered light information (FSC), in particular, forward scattered light signal intensity. Accordingly, generating a first scatter plot based on the scattered light information and the first fluorescence information may include generating a first scatter plot based on the forward scattered light information (FSC), in particular, the forward scattered light signal intensity, and the first fluorescence information (FL1), in particular, the first fluorescence signal intensity, as shown in Figures 2 and 3. Figure 2 illustrates the first scatter plot when the biological sample to be tested is a blood sample, and Figure 3 illustrates the first scatter plot when the biological sample to be tested is a body fluid sample.

[0259] Furthermore, as shown in FIG2 and FIG3 , identifying microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information may include:

[0260] Acquire a microorganism characteristic region P1 and a leukocyte region P2 from the first scattergram, wherein the intensity of the first fluorescence information of the microorganism characteristic region is greater than the intensity of the first fluorescence information of the leukocyte region; and

[0261] The microorganisms in the sample liquid to be tested are identified based on the microorganism characteristic region P1.

[0262] Here, through repeated research by the inventors, in a scatter plot with forward scattered light information as the horizontal coordinate and first fluorescence information as the vertical coordinate, such as the two-dimensional scatter plot shown in Figures 2 and 3, there is a specific area above the white blood cell group (i.e., the white blood cell area) (i.e., the first fluorescence signal intensity of the specific area is not less than the first fluorescence signal intensity of the white blood cell area). When microorganisms are present in the blood sample or body fluid sample, there are always clustered scattered points in the characteristic area, and when microorganisms are not present in the blood sample or body fluid sample, there are no clustered scattered points in the characteristic area. Therefore, the clustered scattered points in the characteristic area can be used to characterize the microbial particle clusters, and the characteristic area can also be called a microbial characteristic area. When the number of scattered points in the characteristic area exceeds a predetermined threshold, it is considered that microorganisms are present in the blood sample or body fluid sample to be tested.

[0263] In some embodiments, identifying microorganisms in the sample liquid based on scattered light information and first fluorescence information may include determining the number of microorganisms in the sample liquid based on the scattered light information, particularly forward scattered light information, and the first fluorescence information. For example, in the embodiments shown in Figures 2 and 3 , the number of scattered points in microbial signature region P1 may be used to indicate the number of microorganisms in the sample liquid.

[0264] In some feasible specific examples, scattered point data (e.g., the number of scattered points in microbial feature region P1) from a large number of biological samples containing microorganisms and the actual number of microorganisms in these biological samples can be pre-collected. A correlation curve between the scattered point data and the actual number of microorganisms can be obtained by fitting, thereby obtaining a corresponding calculation model, such as a linear calculation model. During actual testing of the biological sample to be tested, the microbial scattered point data of the biological sample to be tested is obtained according to the method of the present invention. Based on the microbial scattered point data of the biological sample to be tested and the above-mentioned predetermined calculation model, the number of microorganisms in the biological sample to be tested can be estimated, thereby achieving quantitative analysis of the microorganisms.

[0265] In some embodiments, when the biological sample to be tested is a blood sample to be tested, the sample analysis method 100 may further include: identifying parasites, particularly Plasmodium, in the sample liquid to be tested based on the scattered light information and the first fluorescence information, and in particular identifying parasites in the sample liquid to be tested based on the first scatter plot. This allows for simultaneous detection of microorganisms and parasites in the blood through a single test of the same biological sample to be tested, thereby improving blood testing efficiency and saving reagent costs without increasing blood volume.

[0266] In some embodiments, when the biological sample to be tested is a blood sample, in step S110, the dye reagent may further include a second dye, different from the first dye, capable of staining cells in the blood. In this case, the fluorescence information obtained in step S120 also includes second fluorescence information FL2 from the second dye, particularly the second fluorescence signal intensity. That is, the second fluorescence information includes the fluorescence signal generated by particles in the sample fluid under light excitation after binding with the second dye. Cell parameters of the sample fluid, such as white blood cell parameters and nucleated red blood cell parameters, can thus be further obtained based on the scattered light information and the second fluorescence information.

[0267] As some implementations, a second dye capable of staining white blood cells in the blood, in particular a second dye for classifying white blood cells, is used in step S110. Accordingly, as shown in FIG4 , the sample analysis method 100 may further include step S140: obtaining a white blood cell classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information. In a specific example, the white blood cells in the sample liquid to be tested can be classified into a lymphocyte population, a monocyte population, a neutrophil population, and an eosinophil population based on the scattered light information and the second fluorescence information, and each type of white blood cell is counted to obtain a lymphocyte count and / or a ratio of the lymphocyte count to the white blood cell count, a monocyte count and / or a ratio of the monocyte count to the white blood cell count, a neutrophil count and / or a ratio of the neutrophil count to the white blood cell count, and an eosinophil count and / or a ratio of the eosinophil count to the white blood cell count. In another example, based on the scattered light information and the second fluorescence information, the white blood cells in the sample to be tested can be classified into lymphocytes, monocytes, neutrophils, eosinophils, and basophils, and each type of white blood cell can be counted. This allows for simultaneous microbial detection and white blood cell detection in the blood through a single test of the same blood sample, improving blood testing efficiency and saving reagent costs without increasing blood volume.

[0268] In some embodiments, the second dye used for white blood cell classification can be a nucleic acid dye that can bind to nucleic acid substances in cells, for example, a cyanine cationic compound. For more details, please refer to Chinese patent application CN101750274A filed by the applicant on September 28, 2019, the entire disclosure of which is incorporated herein by reference.

[0269] In some embodiments, the second dye used for leukocyte differentiation is a nucleic acid dye that is not specific for DNA or RNA, for example, including a compound having the following chemical formula II.

[0270]

[0271] Furthermore, the scattered light information may include side scattered light information (SSC) and forward scattered light information (FSC). Accordingly, identifying microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information (i.e., step S130) may include: identifying microorganisms in the sample liquid to be tested based on the forward scattered light information (FSC) and the first fluorescence information (FL1), as shown in FIG5A ; and obtaining a white blood cell classification result for the sample liquid to be tested based on the scattered light information and the second fluorescence information (i.e., step S140) may include: obtaining a white blood cell classification result for the sample liquid to be tested, such as the four-category white blood cell classification result described above, based on the side scattered light information (SSC) and the second fluorescence information (FL2), as shown in FIG5B . For example, a second scatter plot may be generated based on the side scattered light information and the second fluorescence information. On this second scatter plot, the white blood cells in the sample liquid to be tested are classified into a neutrophil population, a lymphocyte population, a monocyte population, and an eosinophil population using a gating technique, and these cell populations are counted.

[0272] Furthermore, step S140 may also include obtaining a white blood cell classification result of the sample liquid to be tested based on the forward scattered light information FSC, the side scattered light information SSC, and the second fluorescence information FL2.

[0273] As another implementation, a second dye that can stain nucleated cells in the blood, such as white blood cells and nucleated red blood cells, is used in step S110. The second dye can be used to identify nucleated red blood cells in particular. Accordingly, as shown in FIG6 , the sample analysis method 100 can also include step S150: obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information, in particular obtaining the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information. In this way, microbial detection in the blood and white blood cell and / or nucleated red blood cell detection can be simultaneously achieved through a single test of the same blood sample to be tested, thereby improving blood testing efficiency and saving reagent costs without increasing the amount of blood used.

[0274] In some embodiments, the second dye used to stain nucleated cells can also be a nucleic acid dye that binds to nucleic acids within the cells. However, unlike the dyes used for white blood cell differentiation described above, it can also be a protein dye that binds to protein within the cells. In one example, the second dye used to stain nucleated cells includes a compound having the following chemical formula III.

[0275] Furthermore, the scattered light information may include forward scattered light information (FSC). Accordingly, identifying microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information (i.e., step S130) may include: identifying the microorganisms in the sample liquid to be tested based on the forward scattered light information (FSC) and the first fluorescence information (FL1), as shown in FIG7A ; and obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information (i.e., step S150) may include: obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the forward scattered light information (FSC) and the second fluorescence information (FL2), as shown in FIG7B .

[0276] In some embodiments, when a dye reagent including a first dye and a second dye is used to treat a biological sample to be tested, in step S120, irradiating the particles flowing through the optical detection area with light includes: irradiating the particles flowing through the optical detection area with light of a single wavelength, especially blue light, for example, blue light with a wavelength of about 450 nanometers.

[0277] Here, when using a single excitation light source to simultaneously excite two dyes, namely a first dye and a second dye, in order to better distinguish and collect first fluorescence information corresponding to the first dye and second fluorescence information corresponding to the second dye, thereby more accurately distinguishing white blood cells and microorganisms in blood using the two dyes under hemolytic conditions, the first dye and the second dye are selected such that the absolute value of the difference between the wavelengths corresponding to the peaks of the emission spectra of the first dye and the second dye is greater than 30 nanometers and less than 80 nanometers. Alternatively or additionally, the first dye and the second dye are selected such that the overlap between the emission spectra of the first dye and the second dye is no more than 50%. By selecting such a first and second dye, the mutual detection interference between the first and second fluorescent signals can be greatly reduced, that is, the differentiation between the first and second fluorescent signals can be greatly increased.

[0278] FIG8 is a schematic diagram illustrating the emission spectra of a first dye and a second dye. The dashed curve represents the emission spectrum 210 of the first dye, and the solid curve represents the emission spectrum 220 of the second dye. The peak of the emission spectrum 210 of the first dye is at point A, and the peak of the emission spectrum 220 of the second dye is at point D. The absolute difference between the abscissas of peak points A and D (i.e., the difference between the wavelengths corresponding to the peaks) is greater than 30 nanometers and less than 80 nanometers. Furthermore, the overlap between the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye can be determined by the ratio of the area of ​​a first polygon to the area of ​​a second polygon. The area of ​​the first polygon is equal to the area of ​​the curved polygon defined by points E, G, and C, while the area of ​​the second polygon is equal to the area of ​​the curved polygon defined by the emission spectrum 210 (or the emission spectrum 220 of the second dye) and a reference line 230. Reference line 230 is a dashed horizontal line parallel to the horizontal axis as shown in FIG8 , located at 5% of the normalized peak values ​​of the emission spectra 210 and 220 of the first dye. Points E and F are the left and right intersection points of the emission spectrum 220 of the second dye and the reference line 230, respectively. Points B and C are the left and right intersection points of the emission spectrum 210 of the first dye and the reference line 230, respectively. Here, the overlap between the emission spectra 210 of the first dye and the emission spectrum 220 of the second dye is no greater than 50%.

[0279] It is further advantageous that, especially when using light of a single wavelength for irradiation, the absolute value of the difference between the wavelengths corresponding to the peaks of the emission spectra of the first dye and the second dye is greater than 40 and less than 80 nanometers, preferably greater than 50 nanometers and less than 80 nanometers, and more preferably greater than 50 nanometers and less than 70 nanometers, thereby further reducing the mutual detection interference between the first fluorescent signal and the second fluorescent signal.

[0280] Furthermore, advantageously, the emission spectra of the first and second dyes overlap by no more than 35%, preferably no more than 15%, thereby further reducing detection interference between the first and second fluorescent signals. The less overlap there is between the emission spectra of the first and second dyes, the easier it is to distinguish the first and second fluorescent signals.

[0281] Next, some embodiments of the first dye of the present invention are described.

[0282] In some embodiments, the first dye may be a large Stokes shift dye. Here, a large Stokes shift dye refers to a dye whose emission spectrum and excitation spectrum have a wavelength difference corresponding to their respective peaks greater than a predetermined threshold.

[0283] Figure 9 is a schematic diagram of the spectrum of a large Stokes shift dye. The excitation spectrum (also called the absorption spectrum) 310 of the large Stokes shift dye is shown by a dashed line, and the emission spectrum 320 is shown by a solid line. The peak point of the excitation spectrum 310 is A1, and the peak point of the emission spectrum 320 is A2. The difference between the abscissas of peak points A2 and A1 (i.e., the difference between the wavelengths corresponding to the peaks of the emission spectrum and the excitation spectrum) is greater than a predetermined threshold. This predetermined threshold can be, for example, greater than 30 nanometers and less than 150 nanometers, preferably greater than 50 nanometers and less than 100 nanometers.

[0284] By using a dye with a large Stokes shift, in particular, mutual detection interference between the first fluorescent signal and the second fluorescent signal can be reduced.

[0285] In some implementations, the first dye is a dye that can specifically bind to deoxyribonucleic acid (ie, DNA), such as a cyanine dye, especially a benzothiazole-type cyanine dye.

[0286] In some embodiments, the first dye may include a compound having a structure of Formula I:

[0287] wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino; R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 alkyl; and Y is absent or a counter anion.

[0288] Here, the first dye proposed in the present invention, which includes a compound having a structure having general formula I, has one or more of the following advantages: good thermal stability; high biological (microorganism, cell) penetrability; can specifically bind to DNA, which is conducive to the specific identification and accurate measurement of DNA; has good permeability to living cells, can enter cells to stain nucleic acids without destroying the cell membrane, has low toxicity and low carcinogenicity; can be excited by blue-green light with a smaller wavelength, thereby being able to identify tiny particles and improving the detection capability of small particles; can use ordinary green semiconductor lasers as light sources, greatly reducing the cost of use; has a simple structure, the raw materials for its preparation are easily available, the synthesis yield is high, and it is easy to achieve industrialization.

[0289] In some embodiments, R1 and R2 are independently selected from C 1-6 Straight chain alkyl, C 1-6 The linear alkylene group -M, M is selected from sulfonic acid group, phenyl group, carboxyl group, mercapto group, and amino group.

[0290] In some embodiments, at least one of R1 and R2 is C 1-18 Straight chain or branched chain alkylene-sulfonic acid group, C 1-18 Straight-chain or branched alkylene-carboxyl.

[0291] In some embodiments, R1 and R2 are different and independently selected from C 1-6 Straight chain alkyl, benzyl, C 1-6 Straight chain alkylene-carboxyl, C 1-6 Straight chain alkylene-sulfonic acid group, C 1-6 Straight chain alkylene-mercapto, C 1-6 In other embodiments, R1 and R2 are the same and are selected from C 1-6 Straight chain alkyl, C 1-6 Straight chain alkylene-sulfonic acid group, C 1-6 Straight chain alkylene-carboxyl.

[0292] In some embodiments, R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 alkyl.

[0293] In some embodiments, when R3 is hydrogen, R1 and R2 are not both methyl and R1 and R2 are not both benzyl.

[0294] In some embodiments, Y in Formula I is a counter anion. In this case, Y can be selected from a halogen ion (e.g., F - 、Cl - Br - , I - )、ClO4 - PF6 - CF3SO3 - 、BF4 - , acetate, methanesulfonate or p-toluenesulfonate. In other embodiments, Y in Formula I is absent, in which case the compound may be an inner salt. "Inner salts" are also referred to as "zwitterions" in the art. For example, the compounds of the present invention may contain both an acidic group (e.g., a sulfonic acid group or a carboxyl group) and a basic group (e.g., an amino group or a thiazole ring) in the molecule, and the acidic and basic groups neutralize each other to form an inner salt.

[0295] It should be understood that when a compound molecule contains multiple acidic groups and / or multiple basic groups, each acidic group and / or each basic group may serve as a salt-forming group. Salts formed by various salt-forming methods are all within the scope of the present invention.

[0296] In some embodiments, the compound of the present invention having the structure of Formula I may have any of the structures shown in the following table:

[0297] Preferably, the compound of the present invention is a compound represented by the above structural formula 5, 6 or 9.

[0298] In other embodiments, in Formula I, R1 and R2 are each independently selected from C 1-18 Alkyl, C 1-18 Sulfonic acid group, C 1-18 Carboxyl, C 1-18 Hydroxyl, C 1-18 NR5R6, benzyl and substituted benzyl, wherein the substituent of the substituted benzyl is selected from C 1-18 Alkyl, CN, COOH, NH2, NO2, OH, SH, C 1-6 Alkoxy, C 1-6 Alkylamino, C 1-6 Amino, halogen and C 1-6 The group consisting of haloalkyl, preferably R1 and R2 are the same C 1-18 Sulfonic acid group; R3 is selected from H, C 1-18 A group consisting of a sulfonic acid group, a phenyl group, OR6 and a halogen; and Y- is a negative ion.

[0299] In some embodiments, the dye reagent of the present invention can be stored in a water-soluble organic phase such as glycerol, ethylene glycol, or the like.

[0300] In some embodiments, the dye reagent of the present invention can be stored alone or mixed with a hemolytic agent.

[0301] The present invention also proposes a technical solution for rapidly detecting parasites in blood based on flow cytometry and fluorescent labeling technology.

[0302] In an embodiment of the present invention, the parasite is selected from the group consisting of Ascaris, Hookworm, Tapeworm, Trichomonas vaginalis, Liver Fluke, Paragonimus westermannii, Toxoplasma, Cysticercus pombe, Trichinella spiralis, Amoeba, Leishmania donovani, Plasmodium, Schistosoma, Filarial worm, Hydatid cyst, Scabies mite, Trichomonas follicularis, Lice, and Flea.

[0303] As shown in FIG10 , an embodiment of the present invention further provides a blood analysis method 1000 for rapidly detecting parasites in blood based on flow cytometry and fluorescent labeling technology. The blood analysis method 1000 includes the following steps S1100 , S1200 , and S1300 .

[0304] In step S1100, a dye reagent containing a first dye and a hemolytic agent for lysing red blood cells are used to treat the same blood sample to obtain a test sample solution. The first dye is capable of staining parasites in the blood. That is, when parasites are present in the blood sample, treating the blood sample with the first dye can stain the parasites. The first dye is a cyanine dye, particularly a benzothiazole-based cyanine dye, and includes a compound having the structure of Formula I:

[0305] wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino, R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Y is absent or is a counter anion.

[0306] For example, in step S1100, a hemolytic agent and a dye reagent may be added sequentially to the same blood sample to obtain a test sample solution, and then the test sample solution may be incubated so that the dye reagent can fully stain the test substance in the test sample solution. For example, in step S1100, the dye reagent may be pre-mixed with the hemolytic agent to obtain a mixed reagent, and then the mixed reagent and the blood sample to be tested may be mixed at a volume ratio of 250:1 to 1000:1. After uniform mixing, the mixed test sample solution may be incubated at a temperature of 25° C. to 50° C. for 10 seconds to 1 minute, preferably for 20 seconds to 40 seconds.

[0307] The various embodiments of the hemolytic agent used in step S110 of the method 100 described above can be correspondingly applied to the hemolytic agent used in step S1100 , and are not described in detail herein.

[0308] The various embodiments of the compound having the structure of Formula I used in step S110 of method 100 described above can be correspondingly applied to the compound of the first dye used in step S1100 , and are not described in detail herein.

[0309] In step S1200, particles in the sample solution are passed through an optical detection zone one by one, and light is applied to the particles passing through the optical detection zone to obtain scattered light information and fluorescence information generated by the particles in the sample solution after being exposed to light. The fluorescence information includes at least first fluorescence information from the first dye, that is, the fluorescence signal generated by the particles in the sample solution after binding to the first dye under light excitation.

[0310] That is, in step S1200, scattered light information and fluorescence information of the sample fluid to be tested are obtained based on the principles of flow cytometry. When light, such as a laser beam, illuminates particles flowing through the optical detection zone, the particle's inherent characteristics (such as volume, staining level, size and content of cellular contents, and nuclear density) can block or redirect the laser beam, thereby generating scattered light at various angles corresponding to the particle's characteristics. This scattered light is received by a signal detector, and optical information related to the particle's structure and composition, i.e., the scattered light information and fluorescence information of the present invention, can be obtained. Here, the scattered light information, for example, includes at least one of forward scattered light information and side scattered light information. Forward scattered light reflects the number and volume of particles, side scattered light (SS) reflects the complexity of the cell's internal structure (such as intracellular granules or nuclei), and fluorescence (FL) reflects the content of nucleic acid in the cell. Using this optical information, various types of particles in the sample fluid to be tested can be identified.

[0311] In an embodiment of the present invention, the scattered light information includes scattered light signal intensity, and the fluorescence information includes fluorescence signal intensity.

[0312] In step S1300 , parasites in the sample liquid are identified based on the scattered light information and the first fluorescence information.

[0313] In some embodiments, step S1300, i.e., identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information, may include: generating a first scattergram based on the scattered light information and the first fluorescence information; and identifying parasites in the sample liquid to be tested based on the first scattergram.

[0314] Furthermore, the scattered light information may include forward scattered light information FSC, in particular, forward scattered light signal intensity. Accordingly, generating a first scatter plot based on the scattered light information and the first fluorescence information may include generating a first scatter plot based on the forward scattered light information FSC, in particular, forward scattered light signal intensity, and the first fluorescence information FL1, in particular, first fluorescence signal intensity, as shown in FIG11 .

[0315] Furthermore, as shown in FIG11 , identifying parasites in the sample liquid based on the scattered light information and the first fluorescence information may include:

[0316] Obtaining a parasite characteristic region P1 and a leukocyte region P2 from the first scattergram, wherein the intensity of the first fluorescence information of the parasite characteristic region is greater than the intensity of the first fluorescence information of the leukocyte region; and

[0317] The parasites in the sample liquid to be tested are identified based on the parasite characteristic region P1.

[0318] Here, through repeated research by the inventors, in a scatter plot with forward scattered light information as the horizontal coordinate and first fluorescence information as the vertical coordinate, such as the two-dimensional scatter plot shown in Figure 11, there is a specific area above the white blood cell group (i.e., the white blood cell area) (i.e., the first fluorescence signal intensity of the specific area is not less than the first fluorescence signal intensity of the white blood cell area). When parasites are present in the blood sample, there are always clustered scattered points in the characteristic area, and when parasites are not present in the blood sample, there are no clustered scattered points in the characteristic area. Therefore, the clustered scattered points in the characteristic area can be used to characterize the parasite particle clusters, and the characteristic area can also be called a parasite characteristic area. When the number of scattered points in the characteristic area exceeds a predetermined threshold, it is considered that parasites are present in the blood sample to be tested.

[0319] In some embodiments, identifying parasites in the sample liquid based on the scattered light information and the first fluorescence information may include determining the number of parasites in the sample liquid based on the scattered light information, particularly the forward scattered light information, and the first fluorescence information. For example, in the embodiment shown in FIG11 , the number of scattered points in the parasite signature region P1 may be used to indicate the number of parasites in the sample liquid.

[0320] In some feasible specific examples, scattered point data (e.g., the number of scattered points in parasite feature region P1) from a large number of blood samples containing parasites and the actual number of parasites in these blood samples can be pre-collected. A correlation curve between the scattered point data and the actual number of parasites can be obtained by fitting, thereby obtaining a corresponding calculation model, such as a linear calculation model. During actual testing of the blood sample to be tested, the parasite scattered point data of the blood sample to be tested is obtained according to the method of the present invention. Based on the parasite scattered point data of the blood sample to be tested and the above-mentioned predetermined calculation model, the number of parasites in the blood sample to be tested can be estimated, thereby achieving quantitative analysis of parasites.

[0321] In some embodiments, the blood analysis method 1000 may further include identifying microorganisms in the test sample based on the scattered light information and the first fluorescence information, particularly identifying microorganisms in the test sample based on the first scatter plot. This allows for simultaneous detection of microorganisms and parasites in the blood through a single test of the same blood sample, thereby improving blood testing efficiency and saving reagent costs without increasing blood volume.

[0322] In some embodiments, the dye reagent of the present invention can be stored in a water-soluble organic phase such as glycerol, ethylene glycol, or the like.

[0323] In some embodiments, the dye reagent of the present invention can be stored alone or mixed with a hemolytic agent.

[0324] In some embodiments, in step S1100, the dye reagent may further include a second dye, different from the first dye, capable of staining cells in blood. In this case, the fluorescence information obtained in step S1200 also includes second fluorescence information FL2 from the second dye, particularly the second fluorescence signal intensity. Specifically, the second fluorescence information includes the fluorescence signal generated by particles in the test sample fluid bound to the second dye under light excitation. Cell parameters of the test sample fluid, such as white blood cell parameters and nucleated red blood cell parameters, can thus be further determined based on the scattered light information and the second fluorescence information.

[0325] As some implementations, a second dye capable of staining nucleated cells in the blood, such as white blood cells and nucleated red blood cells, is used in step S1100. This second dye can be used, in particular, to identify nucleated red blood cells. Accordingly, as shown in FIG12 , the blood analysis method 1000 can further include step S1400: obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information, in particular obtaining the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information. In this way, parasite detection in the blood and white blood cell and / or nucleated red blood cell detection can be simultaneously achieved through a single test of the same blood sample to be tested, thereby improving blood testing efficiency and saving reagent costs without increasing the amount of blood used.

[0326] In some embodiments, the second dye used to stain nucleated cells can be a nucleic acid dye that can bind to nucleic acid substances in the cells, or a protein dye that can bind to protein substances in the cells. In one example, the second dye used to stain nucleated cells includes, for example, a compound having the following chemical formula III.

[0327] Furthermore, the scattered light information may include forward scattered light information (FSC). Accordingly, identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information (i.e., step S1300) may include: identifying parasites in the sample liquid to be tested based on the forward scattered light information (FSC) and the first fluorescence information (FL1), as shown in FIG13A ; and obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information (i.e., step S1400) may include: obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample liquid to be tested based on the forward scattered light information (FSC) and the second fluorescence information (FL2), as shown in FIG13B .

[0328] As another implementation, a second dye capable of staining white blood cells in the blood, particularly a second dye for white blood cell classification, is used in step S1100. Accordingly, as shown in FIG14 , the blood analysis method 1000 may further include step S1500: obtaining a white blood cell classification result for the test sample based on the scattered light information and the second fluorescence information. In a specific example, the white blood cells in the test sample can be classified into a lymphocyte population, a monocyte population, a neutrophil population, and an eosinophil population based on the scattered light information and the second fluorescence information, and each type of white blood cell is counted to obtain a lymphocyte count and / or a ratio of the lymphocyte count to the white blood cell count, a monocyte count and / or a ratio of the monocyte count to the white blood cell count, a neutrophil count and / or a ratio of the neutrophil count to the white blood cell count, and an eosinophil count and / or a ratio of the eosinophil count to the white blood cell count. In another example, based on the scattered light information and the second fluorescence information, the white blood cells in the sample can be classified into lymphocytes, monocytes, neutrophils, eosinophils, and basophils, and each type of white blood cell can be counted. This allows for simultaneous parasite detection and white blood cell detection in the same blood sample, improving blood testing efficiency and saving reagent costs without increasing blood volume.

[0329] In some embodiments, the second dye used for white blood cell classification can also be a nucleic acid dye that can bind to nucleic acid substances in cells, for example, including cyanine cationic compounds. For more details, please refer to Chinese patent application CN101750274A filed by the applicant on September 28, 2019, all of which are incorporated herein by reference.

[0330] In some embodiments, the second dye used for leukocyte differentiation is a nucleic acid dye that is not specific for DNA or RNA, for example, including a compound having the following chemical formula II.

[0331] Furthermore, the scattered light information may include side scattered light information (SSC) and forward scattered light information (FSC). Accordingly, identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information (i.e., step S1300) may include: identifying parasites in the sample liquid to be tested based on the forward scattered light information (FSC) and the first fluorescence information (FL1), as shown in FIG15A ; and obtaining a white blood cell classification result for the sample liquid to be tested based on the scattered light information and the second fluorescence information (i.e., step S1500) may include: obtaining a white blood cell classification result for the sample liquid to be tested, such as the four-category white blood cell classification result described above, based on the side scattered light information (SSC) and the second fluorescence information (FL2), as shown in FIG15B . For example, a second scatter plot may be generated based on the side scattered light information and the second fluorescence information. On this second scatter plot, the white blood cells in the sample liquid to be tested are classified into a neutrophil population, a lymphocyte population, a monocyte population, and an eosinophil population using a gating technique, and these cell populations are counted.

[0332] Furthermore, step S1500 may also include obtaining a white blood cell classification result of the sample liquid to be tested based on the forward scattered light information FSC, the side scattered light information SSC, and the second fluorescence information FL2.

[0333] In some embodiments, when a dye reagent including a first dye and a second dye is used to treat a blood sample to be tested, in step S1200, irradiating the particles flowing through the optical detection area with light includes: irradiating the particles flowing through the optical detection area with light of a single wavelength, especially blue light, for example, blue light with a wavelength of approximately 4500 nanometers.

[0334] Here, when using a single excitation light source to simultaneously excite two dyes, namely, a first dye and a second dye, in order to better distinguish and collect first fluorescence information corresponding to the first dye and second fluorescence information corresponding to the second dye, thereby more accurately distinguishing white blood cells and parasites in blood using the two dyes under hemolytic conditions, the first dye and the second dye are selected such that the absolute value of the difference between the wavelengths corresponding to the peaks of the emission spectra of the first dye and the second dye is greater than 30 nanometers and less than 80 nanometers. Alternatively or additionally, the first dye and the second dye are selected such that the overlap between the emission spectra of the first dye and the second dye is no more than 50%, see Figure 8. By selecting such a first and second dye, the mutual detection interference between the first and second fluorescent signals can be greatly reduced, that is, the differentiation between the first and second fluorescent signals can be greatly increased.

[0335] It is further advantageous that, especially when using light of a single wavelength for irradiation, the absolute value of the difference between the wavelengths corresponding to the peaks of the emission spectra of the first dye and the second dye is greater than 40 and less than 80 nanometers, preferably greater than 50 nanometers and less than 80 nanometers, and more preferably greater than 50 nanometers and less than 70 nanometers, thereby further reducing the mutual detection interference between the first fluorescent signal and the second fluorescent signal.

[0336] Furthermore, advantageously, the emission spectra of the first and second dyes overlap by no more than 35%, preferably no more than 15%, thereby further reducing detection interference between the first and second fluorescent signals. The less overlap there is between the emission spectra of the first and second dyes, the easier it is to distinguish the first and second fluorescent signals.

[0337] In some embodiments, the first dye can be a large Stokes shift dye. A large Stokes shift dye is a dye in which the difference in wavelength between the peak values ​​of the emission spectrum and the excitation spectrum is greater than a predetermined threshold. Referring again to FIG9 , the difference in the abscissa between peak point A2 and peak point A1 is greater than a predetermined threshold. This predetermined threshold can, for example, be greater than 30 nanometers and less than 150 nanometers, preferably greater than 50 nanometers and less than 100 nanometers. The use of a large Stokes shift dye can, in particular, reduce detection interference between the first and second fluorescent signals.

[0338] For other embodiments of the blood analysis method 1000 provided by the present invention, reference may be made to the above description of the sample analysis method 100 .

[0339] The present invention also provides a sample analyzer 400 based on flow cytometry and fluorescent labeling technology. As shown in FIG16 , sample analyzer 400 includes a sampling device 410, a sample preparation device 420, an optical detection device 430, and a processor 440. Sample analyzer 400 also includes a fluidic system (not shown) for connecting sampling device 410, sample preparation device 420, and optical detection device 430 to facilitate fluid transfer between these devices.

[0340] Sampling device 410 is used to quantitatively aspirate a biological sample to be tested, where the biological sample to be tested is a blood sample or a body fluid sample to be tested. For example, sampling device 410 includes a pipette with a pipette nozzle and a drive device for driving the pipette through the pipette nozzle to quantitatively aspirate the biological sample to be tested. The sampling device can transfer the collected biological sample to sample preparation device 420.

[0341] The sample preparation device 420 includes a reaction pool and a reagent supply unit. The reaction pool is used to receive the biological sample to be tested drawn by the sampling device 410, as well as a dye reagent containing a first dye and a hemolytic agent for lysing red blood cells provided by the reagent supply unit. The biological sample to be tested drawn by the sampling device 410, the dye reagent and hemolytic agent provided by the reagent supply unit are mixed in the reaction pool to prepare a sample solution to be tested.

[0342] According to the first embodiment of the present invention, the first dye can stain microorganisms. In this first embodiment, more embodiments of the first dye can refer to the various embodiments of the first dye used in the sample analysis method 100 above, which will not be repeated here.

[0343] According to a second embodiment of the present invention, the first dye is capable of staining parasites and comprises a compound having a structure of general formula I:

[0344] wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino, R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Y is absent or is a counter anion.

[0345] In the second embodiment, more embodiments of the first dye can refer to the various embodiments of the first dye used in the above sample analysis method 100 and the various embodiments of the first dye used in the blood analysis method 1000, which are not repeated here.

[0346] The optical detection device 430 includes a light source, a flow chamber, a scattered light detector and a fluorescence detector. The light source is used to emit a light beam to illuminate the flow chamber. The flow chamber is connected to the reaction pool and particles in the sample liquid to be tested can pass through the flow chamber one by one. The scattered light detector is used to detect scattered light information generated by the particles passing through the flow chamber after being irradiated by light. The fluorescence detector is used to detect fluorescence information generated by the particles passing through the flow chamber after being irradiated by light. The fluorescence information includes first fluorescence information from the first dye.

[0347] As used herein, a flow chamber refers to a chamber containing a focused fluid stream suitable for detecting light scattering and fluorescence signals. When a particle, such as a blood cell, passes through the detection aperture of the flow chamber, the particle scatters an incident light beam directed from a light source into all directions. A light detector can be positioned at one or more different angles relative to the incident light beam to detect the light scattered by the particle, thereby obtaining a light scattering signal. Because different particles have different light scattering properties, light scattering signals can be used to distinguish between different particle populations. Specifically, the light scattering signal detected near the incident light beam is typically referred to as a forward light scattering signal or a low-angle light scattering signal. In some embodiments, the forward light scattering signal can be detected at an angle of approximately 1° to approximately 10° relative to the incident light beam. In other embodiments, the forward light scattering signal can be detected at an angle of approximately 2° to approximately 6° relative to the incident light beam. The light scattering signal detected at a direction approximately 90° relative to the incident light beam is typically referred to as a side light scattering signal. In some embodiments, the side light scattering signal can be detected at an angle of approximately 65° to approximately 115° relative to the incident light beam. Typically, fluorescent signals from blood cells stained with fluorescent dyes are also detected at a direction of approximately 90° to the incident light beam.

[0348] In some embodiments, the optical detection device 430 includes a forward scattered light detector for detecting forward scattered light or a side scattered light detector for detecting side scattered light. The optical detection device 430 preferably includes a forward scattered light detector and a side scattered light detector.

[0349] FIG17 illustrates a specific example of an optical detection device 430. This optical detection device 430 comprises a light source 401, a beam shaping assembly 402, a flow cell 403, and a forward scattered light detector 404, arranged sequentially in a straight line. A dichroic mirror 406 is positioned on one side of the flow cell 403 at a 45° angle to the straight line. A portion of the side light emitted by particles in the flow cell 403 passes through the dichroic mirror 406 and is captured by a fluorescence detector 405, which is positioned behind the dichroic mirror 406 at a 45° angle. Another portion of the side light is reflected by the dichroic mirror 406 and captured by a side scattered light detector 407, which is positioned in front of the dichroic mirror 406 at a 45° angle.

[0350] Processor 440 processes the optical signals collected by optical detection device 430 to obtain desired results. For example, it can generate a two-dimensional or three-dimensional scatter plot based on the various collected optical signals and perform particle analysis on the scatter plot using gating techniques. Processor 440 can also visualize intermediate or final calculation results and display them on display device 450.

[0351] In some embodiments, the processor 440 includes, but is not limited to, a central processing unit (CPU), a microcontroller unit (MCU), a field-programmable gate array (FPGA), a digital signal processor (DSP), and other devices for interpreting computer instructions and processing data in computer software. For example, the processor 440 is configured to execute various computer applications stored in a computer-readable storage medium, thereby enabling the sample analyzer 400 to execute corresponding detection processes and analyze the optical signals detected by the optical detection device 430 in real time.

[0352] The sample analyzer 400 also includes a first housing 460 and a second housing 470. The display device 450 can be, for example, a user interface. The optical detection device 130 and the processor 140 are disposed within the second housing 170. The sample preparation device 420 is, for example, disposed within the first housing 460. The display device 450 is, for example, disposed on the exterior of the first housing 460 and is used to display the test results of the hematology analyzer. In other embodiments, a computer with a display can be remotely connected to the sample analyzer 400, for example, and the computer can be installed in a location remote from the laboratory where the hematology analyzer is located, such as in a doctor's office.

[0353] According to the first embodiment of the present invention, the processor 440 is configured to obtain scattered light information and fluorescence information from the optical detection device 430, and identify microorganisms in the sample liquid based on the scattered light information and the first fluorescence information.

[0354] In some variations of the first embodiment, the processor 440 may be further configured to perform the following steps when identifying microorganisms in the sample liquid based on the scattered light information and the first fluorescence information:

[0355] generating a first scattergram based on the scattered light information and the first fluorescence information; and

[0356] The microorganisms in the sample liquid to be tested are identified based on the first scatter plot.

[0357] In some variations of the first embodiment, the scattered light information includes forward scattered light information. In this case, the processor 440 may be further configured to generate a first scattergram based on the forward scattered light information and the first fluorescence information.

[0358] In some variations of the first embodiment, the processor 440 may be further configured to:

[0359] Acquire a microorganism characteristic region and a leukocyte region from the first scattergram, wherein the intensity of the first fluorescence information of the microorganism characteristic region is greater than the intensity of the first fluorescence information of the leukocyte region; and

[0360] Identify microorganisms in the sample liquid based on microbial characteristic regions.

[0361] In some variations of the first embodiment, the processor 440 may also be configured to perform the following steps when identifying microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information: obtaining the number of microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information.

[0362] In some variations of the first embodiment, the processor 440 may be further configured to identify parasites, especially Plasmodium, in the sample liquid based on the scattered light information and the first fluorescence information.

[0363] In some variations of the first embodiment, the biological sample to be tested is a blood sample, and the dye reagent may further include a second dye, different from the first dye, capable of staining cells in the blood. In this case, the fluorescence information obtained by the optical detection device 430 also includes second fluorescence information from the second dye. Cell parameters of the test sample, such as white blood cell parameters and nucleated red blood cell parameters, can thus be further determined based on the scattered light information and the second fluorescence information.

[0364] FIG18 illustrates another specific example of an optical detection device 430. This optical detection device 430 comprises a laser 431, a frontlight assembly 432, a flow cell 433, a forward scattered light detector 434, a first dichroic mirror 435, a side scattered light detector 436, a second dichroic mirror 437, a first fluorescence detector 438, and a second fluorescence detector 439. The first fluorescence detector 438 is configured to detect a first fluorescence signal corresponding to a first dye generated by particles passing through the flow cell 433 after being irradiated by light, while the second fluorescence detector 439 is configured to detect a second fluorescence signal corresponding to a second dye generated by particles passing through the flow cell 433 after being irradiated by light. Here, the laser 431, the frontlight assembly 432, the flow cell 433, and the forward scattered light detector 434 are sequentially arranged along the optical axis. The frontlight assembly is configured to converge the excitation light emitted by the laser 431 onto the detection zone of the flow cell 433 in the direction of particle flow, thereby causing particles passing through the detection zone of the flow cell 433 to generate scattered light. On one side of flow chamber 433, a first dichroic mirror 435 is arranged at a 45° angle to the optical axis. A portion of the side light generated by particles flowing through the detection zone of flow chamber 433 is reflected by first dichroic mirror 435 and captured by side scattered light detector 436, while another portion of the side light passes through first dichroic mirror 435 and reaches second dichroic mirror 437, which is also arranged downstream of first dichroic mirror 435 at a 45° angle to the optical axis. A portion of the side light that passes through first dichroic mirror 435 is reflected by second dichroic mirror 437 and captured by first fluorescence detector 438, while another portion passes through second dichroic mirror 437 and is captured by second fluorescence detector 439.

[0365] In other embodiments, as shown in FIG19 , unlike the optical detection device shown in FIG18 , the forward scattered light detector 434 can also be arranged obliquely with respect to the optical axis. A reflector 4341 is arranged on the optical axis downstream of the flow chamber along the optical axis to reflect the forward scattered light from the particles into the forward scattered light detector 434, which is arranged obliquely with respect to the optical axis.

[0366] In some variations of the first embodiment, the second dye is a dye capable of staining white blood cells in blood, particularly a dye used for white blood cell classification. Accordingly, the processor 440 may also be configured to obtain a white blood cell classification result of the sample fluid to be tested based on the scattered light information and the second fluorescence information.

[0367] Furthermore, the scattered light information includes side scattered light information and forward scattered light information. Accordingly, the processor 440 can also be configured to: identify microorganisms in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information, and obtain a white blood cell classification result for the sample liquid to be tested based on the side scattered light information and the second fluorescence information.

[0368] In some other variations of the first embodiment, the second dye is a dye capable of staining white blood cells and nucleated red blood cells in blood, and in particular, a dye for identifying nucleated red blood cells. Accordingly, the processor 440 may be further configured to obtain at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the test sample fluid based on the scattered light information and the second fluorescence information.

[0369] Furthermore, the scattered light information includes forward scattered light information. Accordingly, the processor 440 may be further configured to: identify microorganisms in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information, and obtain at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample liquid to be tested based on the forward scattered light information and the second fluorescence information.

[0370] Preferably, the light source can be configured to illuminate the flow chamber with light of a single wavelength. For example, the optical detection device 430 has only one laser source emitting blue-green light, in particular a laser source emitting blue light.

[0371] For more embodiments and advantages of the sample analyzer 400 according to the first embodiment of the present invention, reference may be made to the above description of the sample analysis method 100 of the present invention, which will not be repeated here.

[0372] According to the second embodiment of the present invention, the processor 440 is configured to obtain scattered light information and fluorescence information from the optical detection device 430, and identify parasites in the sample liquid based on the scattered light information and the first fluorescence information.

[0373] In some variations of the second embodiment, the processor 440 may be further configured to perform the following steps when identifying parasites in the sample liquid based on the scattered light information and the first fluorescence information:

[0374] generating a first scattergram based on the scattered light information and the first fluorescence information; and

[0375] Identify parasites in the sample liquid based on the first scatter plot.

[0376] In some variations of the second embodiment, the scattered light information includes forward scattered light information. In this case, the processor 440 may be further configured to generate a first scattergram based on the forward scattered light information and the first fluorescence information.

[0377] In some variations of the second embodiment, the processor 440 may be further configured to:

[0378] Acquire a parasite characteristic region and a leukocyte region from the first scattergram, wherein the intensity of the first fluorescence information of the parasite characteristic region is greater than the intensity of the first fluorescence information of the leukocyte region; and

[0379] Identify parasites in the sample fluid based on parasite characteristic regions.

[0380] In some variations of the second embodiment, the processor 440 may be further configured to perform the following steps when identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information: obtaining the number of parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information.

[0381] In some variations of the second embodiment, the processor 440 may be further configured to identify microorganisms in the sample liquid based on the scattered light information and the first fluorescence information.

[0382] In some variations of the second embodiment, the dye reagent may further include a second dye, different from the first dye, capable of staining cells in blood. In this case, the fluorescence information obtained by the optical detection device 430 also includes second fluorescence information from the second dye. Cell parameters of the sample fluid to be tested, such as white blood cell parameters and nucleated red blood cell parameters, can thus be further determined based on the scattered light information and the second fluorescence information.

[0383] In some variations of the second embodiment, the second dye is a dye capable of staining white blood cells and nucleated red blood cells in blood, and in particular, a dye capable of identifying nucleated red blood cells. Accordingly, the processor 440 may be further configured to obtain at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the test sample based on the scattered light information and the second fluorescence information.

[0384] Furthermore, the scattered light information includes forward scattered light information. Accordingly, the processor 440 may be further configured to: identify parasites in the test sample liquid based on the forward scattered light information and the first fluorescence information, and obtain at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the test sample liquid based on the forward scattered light information and the second fluorescence information.

[0385] In some other variations of the second embodiment, the second dye is a dye capable of staining white blood cells in blood, particularly a dye used for white blood cell classification. Accordingly, the processor 440 may also be configured to obtain a white blood cell classification result of the sample fluid to be tested based on the scattered light information and the second fluorescence information.

[0386] Furthermore, the scattered light information includes side scattered light information and forward scattered light information. Accordingly, the processor 440 may be further configured to: identify parasites in the test sample liquid based on the forward scattered light information and the first fluorescence information, and obtain a white blood cell classification result for the test sample liquid based on the side scattered light information and the second fluorescence information.

[0387] Preferably, the light source can be configured to illuminate the flow chamber with light of a single wavelength. For example, the optical detection device 430 has only one laser source emitting blue-green light, in particular a laser source emitting blue light.

[0388] More embodiments and advantages of the blood analyzer 400 according to the second embodiment of the present invention can be found in the above description of the sample analysis method 100 and the blood analysis method 1000 of the present invention, which will not be repeated here.

[0389] The present invention also proposes the use of DNA-specific dyes (ie dyes that can specifically bind to deoxyribonucleic acid) in identifying microorganisms in a blood sample to be tested using flow cytometry.

[0390] In some embodiments, the DNA-specific dye comprises a compound having the structure of Formula I:

[0391] wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino; R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 alkyl; and Y is absent or a counter anion.

[0392] For more embodiments and advantages of the DNA-specific dye proposed in the present invention, reference may be made to the above description of the sample analysis method 100 of the present invention, which will not be repeated here.

[0393] The present invention also proposes the use of DNA-specific dyes (i.e., dyes that specifically bind to deoxyribonucleic acid) in identifying parasites in blood samples using flow cytometry. The DNA-specific dyes include compounds having the structure of general formula I:

[0394] wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino; R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 alkyl; and Y is absent or a counter anion.

[0395] For more embodiments and advantages of the DNA-specific dye proposed in the present invention, reference may be made to the above description of the sample analysis method 100 and the blood analysis method 1000 of the present invention, which will not be repeated here.

[0396] The present invention will now be described with reference to the following examples which are intended to illustrate the present invention (but not to limit the present invention). Unless otherwise specified, the experiments and methods described in the examples are carried out substantially according to conventional methods well known in the art and described in various references. In addition, where specific conditions are not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Reagents or instruments used that are not specified by the manufacturer are conventional products that can be obtained commercially. It will be appreciated by those skilled in the art that the examples describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0397] Example 1 Synthesis of Compound I

[0398] The structure of compound I can be represented by structural formula 1.

[0399] In the first step, compound (E)-4-2-(5-formyl-2-hydroxyphenylvinyl)benzothiazole-3-butyl-1-sulfonic acid inner salt (structure shown on the right side of reaction formula I) is prepared according to the following reaction formula I.

[0400] To the reaction flask, 5 mL of methanol was added, followed by 1.33 mmol of 4-hydroxyisophthalaldehyde (Shanghai Bidex Pharmaceuticals Co., Ltd.), 0.67 mmol of (E)-4-(2-(5-formyl-2-hydroxyphenyl)benzothiazol-3-yl)-butyl-1-sulfonate, and 2.66 mmol of pyridine (Shanghai Bidex Pharmaceuticals Co., Ltd.). The reaction was stirred at 80°C for 43 hours.

[0401] The reaction mixture was filtered, and the filter cake was washed three times with 10 mL of ethanol. The filter cake was collected and dried under reduced pressure to yield 0.44 mmol of a yellow solid powder, which was (E)-4-(2-(5-formyl-2-hydroxystyryl)benzothiazol-3-yl)-butyl-1-sulfonic acid inner salt. The yield of Reaction Formula I was approximately 33%.

[0402] The compound was subjected to NMR testing and the results are as follows.

[0403] 1H NMR (400MHz, DMSO-d6) δ = 12.04 (s, 1H), 9.95 (s, 1H), 8.78 (d, J = 2.0Hz, 1H), 8.42-8.40 (m, 2H), 8.34-8.24 (m, 2H), 7.94-7.86 ( m,2H),7.80(t,J=7.6Hz,1H),7.18-7.12(m,1H),5.00-4.88(m,2H),2.59–2.56(m,2H),2.08-2.10(m,2H),1.89-1.79(m,2H).

[0404] In the second step, 4-(2-((E)-2-((E)-3-((Z))-2-(3-methylbenzothiazol-2(3H)-enyl)vinyl)-6-oxo-1,4-cyclohexadien-1-yl)vinyl)benzothiazol-3-yl)butanesulfonic acid inner salt (Compound I) was prepared according to the following reaction formula II.

[0405] 5 mL of acetic acid was added to the reaction flask, and 0.44 mmol of (E)-4-(2-(5-formyl-2-hydroxystyryl)benzothiazol-3-yl)-butyl-1-sulfonic acid inner salt obtained in the second step (structure shown on the left side of Reaction Scheme II), 0.72 mmol of 2,3-dimethylbenzothiazolium salt (Shanghai Haohong Biopharmaceutical Technology Co., Ltd.) and 1.58 mmol of sodium acetate were added to the reaction flask in sequence, and the mixture was reacted at 110°C for 12 hours.

[0406] The reaction mixture was filtered, and the filter cake was washed three times with 5 mL of a mixed solution of acetonitrile / water = 1:1 to obtain a crude product.

[0407] The crude product was purified by preparative HPLC to yield approximately 0.01 mmol of a red solid powder, which was 4-(2-((E)-2-((E)-3-((Z))-2-(3-methylbenzothiazol-2(3H)-enyl)vinyl)-6-oxo-1,4-cyclohexadien-1-yl)vinyl)benzothiazol-3-yl)butanesulfonic acid inner salt (Compound I). The yield of Reaction Formula II was approximately 20%.

[0408] The product was subjected to nuclear magnetic resonance testing, and the results were as follows:

[0409] 1H NMR (400MHz, DMSO-d6)δ=8.35-8.14(m,4H),8.09-7.94(m,3H),7.91-7.82(m,2H),7.77-7.74(m,1H),7.70-7.63(m,2H),7.59-7.55(m,1H) ,7.44-7.29(m,1H),6.39(d,J=9.2Hz,1H),4.68-4.66(m,2H),4.15-4.13(m,3H),2.62-2.58(m,2H),2.01-1.92(m,2H),1.85-1.81(m,2H).

[0410] It was verified that the test results were consistent with the structure of Structural Formula 1.

[0411] Example 2 Synthesis of Compound II

[0412] The structure of compound II can be represented by structural formula 2.

[0413] In the first step, 2-methyl-3-(butylsulfonic acid)benzothiazole salt is prepared according to the following reaction formula I (the structure is shown on the right side of reaction formula I).

[0414] 50 mL of toluene was measured in a container, and 34 mmol of benzothiazole (left side of Reaction Scheme 1) and 40 mmol of 1,4-butane sultone (Shanghai Haohong Biopharmaceutical Technology Co., Ltd.) were added to the container. The mixture was refluxed and stirred under nitrogen for 24 hours, after which the reaction was stopped.

[0415] The reaction mixture was filtered, and the filter cake was washed three times with 50 mL of ethyl acetate to obtain a yellow solid product, 2-methyl-3-(butylsulfonic acid)benzothiazolium salt. The yield of reaction formula I was about 62%.

[0416] In the second step, compound II (4-(2-((E)-6-oxo-3-((Z)-2-(3-(4-sulfobutyl)benzo[d]thiazol-2-ylidene)ethylidene)cyclohex-1,4-dien-1-yl)vinyl)benzo[d]thiazol-3-yl)butanesulfonic acid was prepared according to the following reaction formula II.

[0417] 30 mL of acetic acid was placed in a container. 1.7 mmol of 2-methyl-3-(butylsulfonic acid)benzothiazolium salt (structure shown above the arrow in Reaction Scheme II) obtained in the first step, 0.7 mmol of 4-hydroxyisophthalaldehyde, and 2.2 mmol of sodium acetate were added. The reaction was stirred at 80°C under nitrogen for 24 hours.

[0418] The reaction mixture was poured into 50 mL of a pre-prepared solution of petroleum ether:ethyl acetate = 5:1, and the supernatant was decanted to obtain a crude product. The crude product was slurried in 10 mL of acetonitrile and water = 1:1 to obtain approximately 0.34 mmol of a brown solid powder, which was Compound II. The yield of Reaction Formula II was approximately 52%.

[0419] The product was subjected to nuclear magnetic resonance testing, and the results are as follows.

[0420] 1H NMR (400MHz, DMSO-d6, TMS) δ = 8.87 (bs, 1H), 8.42-8.34 (m, 4H), 8.30-8.22 (m, 2H), 8.21-8.07 (m, 3H), 7.88-7. 73(m,4H),7.09(d,J=8.0Hz,1H),5.00-4.95(m,4H),2.68-2.65(m,4H),2.11-2.03(m,4H),1.90-1.84(m,4H).

[0421] It was verified that the test results were consistent with the structure of Structural Formula 2.

[0422] Example 3 Synthesis of Compound VI

[0423] The structure of compound VI can be represented by structural formula 6, wherein Y- is an iodide ion.

[0424] The first step is to prepare 2,3-dimethyl-5-chlorobenzothiazole quaternary ammonium iodide according to the following reaction formula I:

[0425] 20 mL of iodomethane was added to the reaction flask, followed by 10.9 mmol of 5-chloro-2-methylbenzothiazole (Shanghai Bid Pharmaceutical Technology Co., Ltd.). The reaction was heated to 80° C. and stirred at this temperature for 16 hours.

[0426] The reaction mixture was filtered, and the filter cake was washed three times with 10 mL of ethyl acetate. The filter cake was collected and dried under reduced pressure to obtain 7.35 mmol of a white powder solid. This white powder solid was 2,3-dimethyl-5-chlorobenzothiazolium quaternary ammonium iodide (right side of Reaction Scheme 1). The yield of Reaction Scheme 1 was approximately 67%.

[0427] The product was subjected to nuclear magnetic resonance testing, and the results are as follows.

[0428] 1H NMR (400MHz, DMSO-d6) δ = 8.53 (d, J = 12.0Hz, 1H), 8.45 (d, J = 8.8Hz, 1H), 7.88 (dd, J = 2.0, 8.8Hz, 1H), 4.18 (s, 3H), 3.17 (s, 3H)

[0429] In the second step, 5-chloro-2-((E)-2-((E)-3-((Z)-2-(5-chloro-3-methylbenzothiazol-2(3H)-ylidene)vinyl)-6-oxocyclohexa-1,4-dien-1-yl)vinyl-3-methylbenzothiazolium salt (Compound VI) was prepared according to the following reaction formula II:

[0430] 6 mL of acetic acid was placed in a container. 1.67 mmol of 5-chloro-2,3-dimethylbenzothiazole quaternary ammonium salt (structure shown on the left side of Reaction Scheme II) obtained in the first step was added to a mixture of 0.67 mmol of 4-hydroxyisophthalaldehyde (Shanghai Haohong Biopharmaceutical Technology Co., Ltd.) and 2.2 mmol of sodium acetate. The reaction was stirred at 110°C for 12 hours.

[0431] After the reaction is completed, the reaction mixture is filtered and the filter cake is collected to obtain a crude product.

[0432] The crude product was added to 20 mL of acetonitrile and stirred at 90°C for 2 hours. The solid was collected by filtration to obtain approximately 0.46 mmol of a brownish-black solid. This brownish-black solid powder was 5-chloro-2-((E)-2-((E)-3-((Z)-2-(5-chloro-3-methylbenzothiazol-2(3H)-ylidene)vinyl)-6-oxocyclohexa-1,4-dien-1-yl)vinyl-3-methylbenzothiazolium salt (Compound VI). The yield of Reaction Formula II was approximately 70%.

[0433] The product was subjected to nuclear magnetic resonance testing, and the results are as follows.

[0434] 1H NMR (400MHz, DMSO-d6) δ = 8.73 (d, J = 1.2Hz, 1H), 8.53 (d, J = 2.0Hz, 1H), 8.47-8.41 (m, 3H), 8.26-8. 12(m,4H),7.95(d,J=16.4Hz,1H),7.90-7.84(m,2H),7.16(d,J=8.8Hz,1H),4.36(d,J=6.8Hz,6H).

[0435] It was verified that the test results were consistent with the structure of Structural Formula 6.

[0436] Example 4 Synthesis of Compound VII

[0437] The structure of compound VII can be represented by structural formula 7.

[0438] In the first step, 3-(5-carboxylic acid pentyl)-2-methylbenzothiazole bromide is prepared according to the following reaction formula I (the structure is shown on the right side of reaction formula I).

[0439] 67 mmol of benzothiazole (Shanghai Haohong Biopharmaceutical Technology Co., Ltd.) (structure shown on the left side of Reaction Scheme I) was added to a container. 73.72 mmol of 6-bromohexanoic acid was then added. After the reaction system was heated to 140°C, it was stirred for 12 hours. The reaction was then stopped and allowed to cool to room temperature.

[0440] The reaction mixture was filtered, and the filter cake was washed three times with 30 mL of petroleum ether to obtain about 45 mmol of a yellow solid, which was 3-(5-carboxylic acid pentyl)-2-methylbenzothiazole bromide. The yield of reaction formula I was about 78%.

[0441] The product was subjected to nuclear magnetic resonance testing, and the results are as follows.

[0442] 1H NMR (400MHz, DMSO-d6) δ = 8.47 (d, J = 8.0Hz, 1H), 8.35 (d, J = 8.4Hz, 1H), 7.91-7.87 (m, 1H), 7.82-7.78 (m, 1H), 4 .73-4.70(m,2H),3.22(s,3H),2.22(t,J=7.2Hz,2H),1.89-1.78(m,2H),1.60-1.53(m,2H),1.50-1.41(m,2H).

[0443] In the second step, 2-((trans)-2-((trans)-6-oxo-3-((Z)-2-(5-carboxylic acid pentyl)benzothiazol-2(3H)-yl)vinyl)cyclohexa-1,4-dienyl)vinyl-3-(5-carboxylic acid pentyl)benzothiazole salt (Compound VII) was prepared according to the following reaction formula II.

[0444] 1.7 mmol of 3-(5-carboxypentyl)-2-methylbenzothiazolium bromide obtained in the first step and 0.7 mmol of 4-hydroxyisophthalaldehyde were added to 10 mL of acetic acid, followed by 2.2 mmol of sodium acetate. The mixture was stirred at 110°C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and allowed to stand for 16 hours. A large amount of solid precipitated, which was filtered to obtain a crude product.

[0445] The crude product was stirred in 10 mL of acetonitrile for 1 hour and then filtered again. The filter cake was washed three times with 5 mL of acetonitrile. The solid was collected and dried under reduced pressure to obtain about 0.43 mmol of a brown solid powder, which was 2-((trans)-2-((trans)-6-oxo-3-((Z)-2-(5-carboxylic acid pentyl)benzothiazol-2(3H)-yl)vinyl)cyclohexa-1,4-dienyl)vinyl-3-(5-carboxylic acid pentyl)benzothiazole salt (Compound VII). The yield of Reaction Formula II was about 64%.

[0446] The product was subjected to nuclear magnetic resonance testing, and the results are as follows.

[0447] 1H NMR (400MHz, DMSO-d6) δ=12.09-11.94(m,2H),8.76(bs,1H),8.43-8.38(m,3H),8.30(d,J=8.4Hz,1H),8.24-8.20(m,2H),8.16-8.10(m, 2H),7.99-7.95(m,1H),7.89-7.73(m,4H),7.02(d,J=8.8Hz,1H),4.96 -4.95(m,4H),2.23-2.18(m,4H),1.92-1.83(m,4H),1.62-1.47(m,8H).

[0448] It was verified that the test results were consistent with the structure of Structural Formula 7.

[0449] Example 5 Synthesis of Compound VIII

[0450] The structure of compound VIII can be represented by structural formula 8, wherein Y- is an iodide ion.

[0451] In the first step, 2,3-dimethyl-5-fluorobenzothiazole quaternary ammonium iodide is prepared according to the following reaction formula I (the structure is shown on the right side of reaction formula I).

[0452] 10 mL of iodomethane was added to the reaction flask, followed by 9.0 mmol of 5-fluoro-2-methylbenzothiazole (Shanghai Haohong Biopharmaceutical Technology Co., Ltd.), and the reaction was heated to 80° C. and stirred at this temperature for 12 hours.

[0453] The reaction mixture was filtered, and the filter cake was washed three times with 10 mL of ethyl acetate. The filter cake was collected and dried under reduced pressure to yield 7.1 mmol of a white powder solid B. This white powder solid was 2,3-dimethyl-5-fluorobenzothiazolyl quaternary ammonium iodide (structure shown on the right side of Reaction Scheme I). The yield of Reaction Scheme I was approximately 79%.

[0454] The product was subjected to nuclear magnetic resonance testing, and the results are as follows.

[0455] H NMR (400MHz, DMSO-d6) δ = 8.50 (dd, J = 5.2, 9.2Hz, 1H), 8.34 (dd, J = 2.4, 8.4Hz, 1H), 7.74 (dt, J = 2.0, 8.8Hz, 1H), 4.18 (s, 3H), 3.18 (s, 3H).

[0456] It has been verified that the test results are consistent with the product structure of reaction formula I.

[0457] In the second step, 5-fluoro-2-((E)-2-((E)-3-((Z)-2-(5-fluoro-3-methylbenzothiazol-2(3H)-ylidene)vinyl)-6-oxocyclohexa-1,4-dien-1-yl)vinyl-3-methylbenzothiazolium salt (Compound VIII) is prepared according to the following reaction formula II:

[0458] 2 mmol of 2,3-dimethyl-5-fluorobenzothiazolyl quaternary ammonium iodide (structure shown on the left side of Reaction Scheme II) obtained in the first step and 0.67 mmol of 4-hydroxyisophthalaldehyde were added to 10 mL of acetic acid, followed by 2.2 mmol of sodium acetate. The reaction was stirred at 120°C for 12 hours.

[0459] After the reaction is completed, the reaction mixture is filtered, the filter cake is washed three times with 10 mL of acetonitrile, and the solid is collected to obtain approximately 0.46 mmol of a brown-black solid. The brown-black solid powder is 5-fluoro-2-((E)-2-((E)-3-((Z)-2-(5-fluoro-3-methylbenzothiazol-2(3H)-ylidene)vinyl)-6-oxocyclohexa-1,4-dien-1-yl)vinyl-3-methylbenzothiazolium salt (Compound VIII), and the yield of Reaction Formula II is approximately 95%.

[0460] The product was subjected to nuclear magnetic resonance testing, and the results are as follows.

[0461] 1H NMR (400MHz, DMSO-d6) δ = 8.72 (bs, 1H), 8.48-8.18 (m, 8H), 7.97-7.94 (m, 1H), 7.73 (bs, 2H), 7.15 (bs, 1H), 4.35 (bs, 6H).

[0462] It was verified that the test results were consistent with the structure of structural formula 8.

[0463] Example 6 Synthesis of Compound IX

[0464] The structure of compound IX can be represented by structural formula 9, wherein Y- is an iodide ion.

[0465] In the first step, 2-methyl-5-cyanobenzothiazole is prepared according to the following reaction formula I (the structure is shown on the right side of reaction formula I).

[0466] To a reaction flask, add 55 mL of deionized water. Under a nitrogen atmosphere, add and dissolve 0.29 mmol of potassium acetate. Then, add 55 mL of dioxane, 5.7 mmol of 5-bromo-2-methylbenzothiazole (Shanghai Bid Pharmaceutical Technology Co., Ltd.), 2.85 mmol of potassium ferricyanide trihydrate, and 0.57 mmol of XPhos-Pd-G3. After all the raw materials and reagents have been added, replace the atmosphere with nitrogen. The reaction mixture is reacted at 100°C for 1 hour.

[0467] After the reaction, the mixture was diluted with 60 mL of water and extracted with 200 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate for 30 min, filtered, and the filtrate was concentrated under reduced pressure to yield a crude yellow oil. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1 to 5:1) to yield 5.72 mmol of a gray powdery solid, 2-methyl-5-cyanobenzothiazole (see the structure on the right side of Reaction Scheme 1).

[0468] The product was subjected to nuclear magnetic resonance testing, and the results are as follows.

[0469] 1H NMR (400MHz, DMSO-d6) δ = 8.44 (d, J = 1.2Hz, 1H), 8.28 (d, J = 8.6Hz, 1H), 7.79 (dd, J = 1.6, 8.4Hz, 1H), 2.85 (s, 3H).

[0470] It has been verified that the test results are consistent with the product structure of reaction formula I.

[0471] In the second step, 2,3-dimethyl-5-cyanobenzothiazole quaternary ammonium iodide is prepared according to the following reaction formula II (the structure is shown on the right side of reaction formula II).

[0472] 20 mL of iodomethane was added to the reaction flask, followed by 5.7 mmol of 5-cyano-2-methylbenzothiazole (Chengdu Forest Technology Development Co., Ltd.), and the reaction was heated to 80° C. and stirred at this temperature for 16 hours.

[0473] The reaction mixture was filtered, and the filter cake was washed three times with 10 mL of ethyl acetate. The filter cake was collected and dried under reduced pressure to yield 2.18 mmol of a yellow powdery solid, which was 2,3-dimethyl-5-cyanobenzothiazolyl quaternary ammonium iodide (structure shown on the right side of Reaction Scheme II). The yield of Reaction Scheme II was approximately 38%.

[0474] The product was subjected to nuclear magnetic resonance testing, and the results are as follows.

[0475] 1H NMR (400MHz, DMSO-d6) δ = 8.98 (s, 1H), 8.63 (d, J = 8.8Hz, 1H), 8.22 (dd, J = 2.0, 8.8Hz, 1H), 4.22 (s, 3H), 3.21 (s, 3H).

[0476] It has been verified that the test results are consistent with the product structure of Reaction Formula II.

[0477] In the third step, 2-((trans)-2-((trans)-6-oxo-3-((Z)-2-(5-cyanobenzothiazol-2(3H)-yl)vinyl)cyclohexa-1,4-dienyl)vinyl-3-(5-cyano)benzothiazole salt (Compound IX) was prepared according to the following reaction formula III:

[0478] Measure 30 mL of acetic acid into a container, then add 2 mmol of the 5-cyano-2,3-dimethylbenzothiazole quaternary ammonium salt (structure shown on the left side of Reaction Scheme III) obtained in the first step, 0.67 mmol of 4-hydroxyisophthalaldehyde, and 2.2 mmol of sodium acetate. Stir the reaction at 120°C under nitrogen for 12 hours.

[0479] The reaction mixture was filtered, and the filter cake was washed with acetonitrile, and the filter cake was collected to obtain a crude product.

[0480] The crude product was repeatedly slurried three times with 10 mL of acetonitrile to obtain approximately 0.37 mmol of a brown-black solid. The brown-black solid powder was 2-((trans)-2-((trans)-6-oxo-3-((Z)-2-(5-cyanobenzothiazol-2(3H)-yl)vinyl)cyclohexa-1,4-dienyl)vinyl-3-(5-cyano)benzothiazole salt (Compound IX). The yield of Reaction Formula III was approximately 52%.

[0481] The product was subjected to nuclear magnetic resonance testing, and the results are as follows.

[0482] 1H NMR (400MHz, DMSO-d6) δ = 8.95-8.87 (m, 2H), 8.66-8.60 (m, 3H), 8.30-8.15 (m, 6H), 7.93-7.89 (m, 1H), 7.11 (d, J = 8.0Hz, 1H), 4.36-4.34 (m, 6H).

[0483] It was verified that the test results were consistent with the structure of structural formula 9.

[0484] Example 7 Evaluation of dye specificity

[0485] The specificity of the dye was evaluated by measuring the fluorescence intensity change of dye compound II with increasing calf thymus DNA and RNA concentrations and the linear relationship between the maximum fluorescence emission peak intensity and the calf thymus DNA and RNA concentrations.

[0486] Prepare a calf thymus DNA aqueous solution at a specific concentration and measure its absorbance at 260 nm using a UV spectrophotometer to calibrate the concentration to 1.8 mM. Take 100 μL of calf thymus DNA calibrated to 1.8 mM and add 290 μL of water to dilute the solution to a 0.5 mM calf thymus DNA aqueous solution. Prepare a 1 mM solution of compound B in DMSO (dimethyl sulfoxide), take 1.5 μL of the solution, add M-60LN hemolytic reagent (Mindray), and add the solution to 3 mL. Place the solution in a cuvette and measure its fluorescence intensity. Subsequently, add 0.6 μL of the 0.5 mM calf thymus DNA aqueous solution to the cuvette, stir the buffer solution thoroughly, and incubate at 37°C for 3 minutes before measuring the fluorescence intensity. The final calf thymus DNA concentration in the cuvette is 1 μM. Calculate the intensity at the maximum fluorescence emission peak for each calf thymus DNA concentration and plot a linear relationship between fluorescence intensity and calf thymus DNA concentration. The linear relationship between RNA concentration and fluorescence intensity was also tested using the same procedure described above. The instruments used were a UV-visible spectrophotometer, model: HP8453; and a fluorescence spectrophotometer, model: FP-6500.

[0487] Figure 20 shows the changes in the fluorescence spectrum of Compound II with increasing DNA concentration. Figure 21 shows the changes in the fluorescence spectrum of Compound II with increasing RNA concentration. Figure 22 is a graph showing the linear relationship between fluorescence intensity and calf thymus DNA and RNA concentrations.

[0488] As can be seen from the figure, compound II has a concentration-dependent relationship with DNA, but has no concentration-dependent relationship with RNA, indicating that compound II can specifically bind to DNA.

[0489] Example 8 Observation of the staining of HepG2 living cells by compound II under laser microscope

[0490] Add 10 μL of PBS buffer containing Compound II at a concentration of 1 mM to a six-well plate containing HepG2 cells. Incubate in a cell culture incubator at 37°C and 5% CO₂ for 30 minutes. Then, wash three times with PBS and add cell culture medium. Cell morphology was observed using a confocal laser scanning microscope (FV1000IX81, Japan).

[0491] Figure 23 shows the observation results. The center image is a whitefield micrograph of Compound II staining live HepG2 cells. The left image is a fluorescence micrograph of Compound II staining live HepG2 cells. The right image is an overlay of the brightfield and fluorescence images. As can be seen, Compound II clearly stains the HepG2 cell nuclei, demonstrating the dye's good permeability and robust nucleic acid staining ability.

[0492] Example 9 Observation of the staining of HepG2 living cells by compound III under laser microscope

[0493] Add 10 μL of PBS buffer containing Compound III at a concentration of 1 mM to a six-well plate containing HepG2 cells. Incubate in a cell culture incubator at 37°C and 5% CO₂ for 30 minutes. Then, wash three times with PBS and add cell culture medium. Cell morphology was observed using a confocal laser scanning microscope (FV1000IX81, Japan).

[0494] Figure 24 shows the observation results. The center image is a whitefield micrograph of live HepG2 cells stained with Compound III. The left image is a fluorescence micrograph of live HepG2 cells stained with Compound III. The right image is an overlay of the brightfield and fluorescence images. As can be seen, Compound III clearly stains the HepG2 cell nuclei, demonstrating the dye's good permeability and robust nucleic acid staining ability.

[0495] Example 10 Evaluation of dye stability

[0496] Dyes used in commercial cell dyes must have a certain degree of high-temperature stability. Existing dyes have limited commercial applications due to their poor high-temperature stability. To improve this situation, the present invention has developed a variety of new dyes designed from the perspective of molecular structure to improve dye stability.

[0497] To evaluate high-temperature stability, 50 mg / L solutions of different dyes in ethylene glycol were placed in a 50°C incubator. 0.5 ml of the solution was sampled at various times to determine the dye concentration. Degradation curves were plotted based on the dye concentration at different time points. The dyes tested included QCy-DT, dye 5 (Compound V), dye 6 (Compound VI), and dye 9 (Compound IX), all reported in the literature (Nucleic Acids Research, 2015, Vol. 43, No. 18, 8651–8663). The instrument used was a UV-VIS spectrophotometer (Model TCC-240A, Shimadzu, Japan).

[0498] Figure 25 shows the degradation curves. The four curves, from top to bottom, correspond to Dye 5, Dye 6, Dye 9, and Dye QCy-DT. As can be seen from the figure, QCy-DT suffers the most severe degradation, while Dyes 5, Dye 6, and Dye 9 show less degradation. This suggests that the introduction of an electron-withdrawing group at R3 in the compound can improve its stability to a certain extent, facilitating its commercial application, such as blood cell staining.

[0499] Example 11 Evaluation of dye cell penetration

[0500] Dyes used in commercial cell dyes must possess good cell penetration. Existing dyes have low molecular structure logP values ​​(the logarithm of the ratio of the compound's partition coefficient between n-octanol and water), resulting in poor penetration into living cells and limited commercial application. To address this issue, the present invention incorporates a molecular structure design that incorporates multiple chemical groups, enhancing both dye stability and cell penetration.

[0501] To verify cell penetration, 10 μL of PBS buffer containing different dye compounds at a 1 mM concentration was added to a 12-well plate containing cultured HepG2 cells and incubated in a cell culture incubator at 37°C and 5% CO2. The plates were then washed three times with PBS and then re-added with cell culture medium at different time points. The fluorescence of each well was measured using a multi-function microplate reader (Thermo, USA).

[0502] Figure 26 shows the test results. The four curves, from top to bottom, correspond to Dye 6, Dye 9, Dye 5, and Dye QCy-DT. As shown in the figure, Dyes 5, 6, and 9 have better cell penetration than QCy-DT.

[0503] Example 12 Use of the cyanine dye of the present invention in identifying microorganisms in a blood sample using flow cytometry

[0504] First, prepare dye reagent A1 and hemolytic agent B1 according to the following formula.

[0505] The first dye includes a compound having the above structural formula 1 and is used to dye microorganisms, and the second dye includes a compound having the following chemical formula and is used to dye cells:

[0506] Then, 20 microliters of dye reagent A1, 1 milliliter of hemolytic agent B1 and 20 microliters of the anticoagulated blood sample containing Klebsiella pneumoniae were mixed, and the mixture was incubated at 42°C for 30 seconds to form a sample solution for measurement. Next, the sample solution was tested using a flow cytometer with a blue laser having an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2. A first scatter plot as shown in FIG27A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in FIG27B was generated based on the side scattered light intensity information and the second fluorescence intensity. Based on the first scatter plot shown in FIG27A, the presence of microorganisms in the blood sample to be tested can be identified, and based on the second scatter plot shown in FIG27B, the white blood cells in the blood sample to be tested can be classified into four categories, and the white blood cell classification results shown in Table 1 were obtained.

[0507] The same blood sample was tested on an existing blood analyzer (Mindray, model BC-6800) using the DIFF channel (using the DIFF reagents provided with Mindray BC-6800), and the white blood cell classification results shown in Table 1 were obtained.

[0508] Table 1 Leukocyte classification results

[0509] As can be seen from Table 1, the white blood cell classification results obtained according to the present invention are basically consistent with those obtained using the existing BC-6800. Therefore, it can be seen that the embodiments of the present invention can simultaneously detect microorganisms and white blood cells in the blood through a single test of the same blood sample.

[0510] Example 13 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples using flow cytometry

[0511] A certain amount of Pseudomonas Klebsiellae is added to physiological saline to obtain a simulated body fluid sample to be tested; the above-mentioned simulated body fluid sample to be tested is tested using the reagents and methods of Example 12 to obtain the forward scattered light intensity FSC and the first fluorescence intensity FL1; a first scatter plot as shown in Figure 28 is generated based on the forward scattered light intensity information and the first fluorescence intensity; based on the first scatter plot shown in Figure 28, the presence of microorganisms in the simulated body fluid sample can be identified.

[0512] Example 14 Use of the cyanine dye of the present invention in identifying microorganisms in a blood sample using flow cytometry

[0513] First, prepare dye reagent A2 and hemolytic agent B2 according to the following formula.

[0514] The first dye includes a compound having the above structural formula 2 and is used to stain microorganisms, and the second dye includes a compound having the following chemical formula and is used to stain cells:

[0515] Then, 20 microliters of dye reagent A2, 1 milliliter of hemolytic agent B2 and 20 microliters of the anticoagulated blood sample containing Klebsiella pneumoniae were mixed, and the mixture was incubated at 42°C for 30 seconds to form a sample solution for measurement. Next, the sample solution was tested using a flow cytometer with a blue laser having an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2. A first scatter plot as shown in FIG29A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in FIG29B was generated based on the side scattered light intensity information and the second fluorescence intensity. Based on the first scatter plot shown in FIG29A , the presence of microorganisms in the blood sample to be tested can be identified, and based on the second scatter plot shown in FIG29B , the white blood cells in the blood sample to be tested can be classified into four categories, and the white blood cell classification results shown in Table 2 were obtained.

[0516] The same blood sample was tested on an existing blood analyzer (Mindray, model BC-6800) using the DIFF channel (using the DIFF reagents provided with Mindray BC-6800), and the white blood cell classification results shown in Table 2 were obtained.

[0517] Table 2 Leukocyte classification results

[0518] As can be seen from Table 2, the white blood cell classification results obtained according to the present invention are basically consistent with those obtained according to the existing BC-6800. Therefore, it can be seen that the embodiments of the present invention can simultaneously detect microorganisms and white blood cells in the blood through a single test of the same blood sample.

[0519] Example 15 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples using flow cytometry

[0520] A certain amount of Pseudomonas Klebsiellae is added to physiological saline to obtain a simulated body fluid sample to be tested; the simulated body fluid sample to be tested is tested using the reagents and methods of Example 14 to obtain forward scattered light intensity FSC and first fluorescence intensity FL1; a first scatter plot as shown in Figure 30 is generated based on the forward scattered light intensity information and the first fluorescence intensity; based on the first scatter plot shown in Figure 11, the presence of microorganisms in the simulated body fluid sample can be identified.

[0521] Example 16 Use of the cyanine dye of the present invention in identifying microorganisms in a blood sample using flow cytometry

[0522] First, prepare dye reagent A3 and hemolytic agent B3 according to the following formula.

[0523] The first dye includes a compound having the above structural formula 3 and is used to dye microorganisms, and the second dye includes a compound having the following chemical formula and is used to dye cells:

[0524] Then, 20 microliters of dye reagent A3, 1 milliliter of hemolytic agent B3 and 20 microliters of the anticoagulant-treated blood sample containing Escherichia coli were mixed, and the mixture was incubated at 42°C for 30 seconds to form a sample solution for measurement; then, the sample solution was tested using a flow analyzer with a blue laser having an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; a first scatter plot as shown in FIG31A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in FIG31B was generated based on the side scattered light intensity information and the second fluorescence intensity; based on the first scatter plot shown in FIG31A, the presence of microorganisms in the blood sample to be tested can be identified, and based on the second scatter plot shown in FIG31B, the white blood cells in the blood sample to be tested can be classified into four categories, and the white blood cell classification results shown in Table 3 are obtained.

[0525] The same blood sample was tested on an existing blood analyzer (Mindray, model BC-6800) using the DIFF channel (using the DIFF reagents provided with Mindray BC-6800), and the white blood cell classification results shown in Table 3 were obtained.

[0526] Table 3 Leukocyte classification results

[0527] As can be seen from Table 3, the white blood cell classification results obtained according to the present invention are basically consistent with those obtained according to the existing BC-6800. This shows that the embodiment of the present invention can simultaneously detect microorganisms and white blood cells in the blood through a single test of the same blood sample.

[0528] Example 17 Use of the cyanine dye of the present invention in identifying microorganisms in a simulated body fluid sample using flow cytometry

[0529] A certain amount of Escherichia coli is added to physiological saline to obtain a simulated body fluid sample to be tested; the above-mentioned simulated body fluid sample to be tested is tested using the reagents and methods of Example 16 to obtain the forward scattered light intensity FSC and the first fluorescence intensity FL1; a first scatter plot as shown in Figure 32 is generated based on the forward scattered light intensity information and the first fluorescence intensity; based on the first scatter plot shown in Figure 32, the presence of microorganisms in the simulated body fluid sample can be identified.

[0530] Example 18 Use of the cyanine dye of the present invention in identifying microorganisms in a blood sample using flow cytometry

[0531] First, prepare dye reagent A4 and hemolytic agent B4 according to the following formula.

[0532] The first dye includes a compound having the above structural formula 1 and is used to dye microorganisms, and the second dye includes a compound having the following chemical formula and is used to dye cells:

[0533] Then, 20 microliters of dye reagent A4, 1 milliliter of hemolytic agent B4 and 20 microliters of the anticoagulated blood sample containing Klebsiella pneumoniae were mixed, and the mixture was incubated at 42°C for 30 seconds to form a sample solution for measurement; then, the sample solution was tested using a flow analyzer with a blue laser having an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, a first fluorescence intensity FL1 and a second fluorescence intensity FL2; a first scatter plot as shown in Figure 33A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in Figure 33B was generated based on the forward scattered light intensity information and the second fluorescence intensity; based on the first scatter plot shown in Figure 33A, the presence of microorganisms in the blood sample to be tested can be identified, and based on the second scatter plot shown in Figure 33B, the white blood cells, nucleated red blood cells and basophils in the blood sample to be tested can be identified and counted.

[0534] It can be seen that the embodiment of the present invention can simultaneously detect microorganisms and nucleated red blood cells in the blood through a single test of the same blood sample.

[0535] Example 19 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples using flow cytometry

[0536] A certain amount of Pseudomonas Klebsiellae is added to physiological saline to obtain a simulated body fluid sample to be tested; the above-mentioned simulated body fluid sample to be tested is tested using the reagents and methods of Example 18 to obtain the forward scattered light intensity FSC and the first fluorescence intensity FL1; a first scatter plot as shown in Figure 34 is generated based on the forward scattered light intensity information and the first fluorescence intensity; based on the first scatter plot shown in Figure 34, the presence of microorganisms in the simulated body fluid sample can be identified.

[0537] Example 20 Use of the cyanine dye of the present invention in identifying microorganisms in a blood sample using flow cytometry

[0538] First, prepare dye reagent A5 and hemolytic agent B5 according to the following formula.

[0539] The first dye includes a compound having the above structural formula 3 and is used to dye microorganisms, and the second dye includes a compound having the following chemical formula and is used to dye cells:

[0540] Then, 20 microliters of dye reagent A5, 1 milliliter of hemolytic agent B5 and 20 microliters of the anticoagulated blood sample containing Klebsiella pneumoniae were mixed, and the mixture was incubated at 42°C for 30 seconds to form a sample solution for measurement; then, the sample solution was tested using a flow analyzer with a blue laser having an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, a first fluorescence intensity FL1 and a second fluorescence intensity FL2; a first scatter plot as shown in Figure 35A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in Figure 35B was generated based on the forward scattered light intensity information and the second fluorescence intensity; based on the first scatter plot shown in Figure 35A, the presence of microorganisms in the blood sample to be tested can be identified, and based on the second scatter plot shown in Figure 35B, the white blood cells, nucleated red blood cells and basophils in the blood sample to be tested can be identified and counted.

[0541] It can be seen that the embodiment of the present invention can simultaneously detect microorganisms and nucleated red blood cells in the blood through a single test of the same blood sample.

[0542] Example 21 Use of the cyanine dye of the present invention in identifying microorganisms in a simulated body fluid sample using flow cytometry

[0543] A certain amount of Pseudomonas Klebsiella is added to physiological saline to obtain a simulated body fluid sample to be tested; the above-mentioned simulated body fluid sample to be tested is tested using the reagents and methods of Example 20 to obtain the forward scattered light intensity FSC and the first fluorescence intensity FL1; a first scatter plot as shown in Figure 36 is generated based on the forward scattered light intensity information and the first fluorescence intensity; based on the first scatter plot shown in Figure 36, the presence of microorganisms in the simulated body fluid sample can be identified.

[0544] Example 22 Use of the cyanine dye of the present invention in identifying parasites in a blood sample using flow cytometry

[0545] 20 μL of dye reagent A4 (Example 18, a first dye for staining parasites), 1 mL of hemolytic agent B4 (Example 18), and 20 μL of an anticoagulated blood sample from a patient infected with malaria were mixed, and the mixture was incubated at 42° C. for 30 seconds to form a test sample solution for measurement. Next, the test sample solution was tested using a flow cytometer with a blue laser having an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, a first fluorescence intensity FL1, and a second fluorescence intensity FL2. A first scatter plot as shown in FIG. 37A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in FIG. 37B was generated based on the forward scattered light intensity information and the second fluorescence intensity. The presence of malarial parasites in the test blood sample can be identified based on the first scatter plot shown in FIG. 37A , and the white blood cells, nucleated red blood cells, and basophils in the test blood sample can be identified and counted based on the second scatter plot shown in FIG.

[0546] It can be seen that the embodiment of the present invention can simultaneously detect parasites and nucleated red blood cells in the blood through a single test of the same blood sample.

[0547] Example 23 Use of the cyanine dye of the present invention in identifying parasites in a blood sample using flow cytometry

[0548] First, prepare dye reagent A6 and hemolytic agent B6 according to the following formula.

[0549] The first dye includes a compound having the above structural formula 2 and is used to dye parasites, and the second dye includes a compound having the following chemical formula and is used to dye cells:

[0550] Then, 20 microliters of dye reagent A6, 1 milliliter of hemolytic agent B6 and 20 microliters of the anticoagulated blood sample of the patient infected with malaria were mixed, and the mixture was incubated at 42°C for 30 seconds to form a sample solution for measurement; then, the sample solution was tested using a flow analyzer with a blue laser with an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; a first scatter plot as shown in Figure 38A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in Figure 38B was generated based on the forward scattered light intensity information and the second fluorescence intensity; based on the first scatter plot shown in Figure 38A, the presence of malarial parasites in the blood sample could be identified, and based on the second scatter plot shown in Figure 38B, the white blood cells, nucleated red blood cells and basophils in the blood sample could be identified and counted.

[0551] It can be seen that the embodiment of the present invention can simultaneously detect parasites and nucleated red blood cells in the blood through a single test of the same blood sample.

[0552] Example 24 Use of the cyanine dye of the present invention in identifying parasites in a blood sample using flow cytometry

[0553] 20 μL of dye reagent A5 (Example 20, the first dye for staining parasites), 1 mL of hemolytic agent B5 (Example 20) were mixed with 20 μL of an anticoagulated blood sample from a patient infected with malaria, and the mixture was incubated at 42° C. for 30 seconds to form a test sample solution for measurement. Next, the test sample solution was tested using a flow cytometer with a blue laser having an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, first fluorescence intensity FL1, and second fluorescence intensity FL2. A first scatter plot as shown in FIG. 39A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in FIG. 39B was generated based on the forward scattered light intensity information and the second fluorescence intensity. The presence of malarial parasites in the test blood sample can be identified based on the first scatter plot shown in FIG. 39A , and the white blood cells, nucleated red blood cells, and basophils in the test blood sample can be identified and counted based on the second scatter plot shown in FIG.

[0554] It can be seen that the embodiment of the present invention can simultaneously detect parasites and nucleated red blood cells in the blood through a single test of the same blood sample.

[0555] Example 25 Use of the cyanine dye of the present invention in identifying parasites in a blood sample using flow cytometry

[0556] 20 μL of dye reagent A2 (Example 14, the first dye is used to stain parasites), 1 mL of hemolytic agent B2 (Example 14) and 20 μL of an anticoagulated blood sample from a patient infected with malaria were mixed, and the mixture was incubated at 42° C. for 30 seconds to form a test sample solution for measurement. Next, the test sample solution was tested using a flow cytometer with a blue laser having an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2. A first scatter plot as shown in FIG40A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in FIG40B was generated based on the side scattered light intensity information and the second fluorescence intensity. Based on the first scatter plot shown in FIG40A , the presence of malarial parasites in the test blood sample can be identified, and based on the second scatter plot shown in FIG40B , the white blood cells in the test blood sample can be classified into four categories, resulting in the white blood cell classification results shown in Table 4.

[0557] The same blood sample was tested on an existing blood analyzer (Mindray, model BC-6800) using the DIFF channel (using the DIFF reagents provided with Mindray BC-6800), and the white blood cell classification results shown in Table 4 were obtained.

[0558] Table 4 Leukocyte classification results

[0559] As can be seen from Table 4, the white blood cell classification results obtained according to the present invention are substantially consistent with those obtained using the existing BC-6800. This shows that the embodiments of the present invention can simultaneously detect parasites and white blood cells in the blood through a single test of the same blood sample.

[0560] Example 26 Use of the cyanine dye of the present invention in identifying parasites in a blood sample using flow cytometry

[0561] First, prepare dye reagent A7 and hemolytic agent B7 according to the following formula.

[0562] The first dye includes a compound having the structural formula 3 in Table 1 and is used to stain parasites, and the second dye includes a compound having the following chemical formula and is used to stain cells:

[0563] Then, 20 microliters of dye reagent A7, 1 milliliter of hemolytic agent B7 and 20 microliters of the anticoagulated blood sample of the patient infected with malaria were mixed, and the mixture was incubated at 42°C for 30 seconds to form a sample solution for measurement; then, the sample solution was tested using a flow analyzer with a blue laser with an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; a first scatter plot as shown in Figure 41A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in Figure 41B was generated based on the side scattered light intensity information and the second fluorescence intensity; based on the first scatter plot shown in Figure 41A, the presence of malarial parasites in the blood sample to be tested can be identified, and based on the second scatter plot shown in Figure 41B, the white blood cells in the blood sample to be tested can be classified into four categories, and the white blood cell classification results shown in Table 5 are obtained.

[0564] The same blood sample was tested on an existing blood analyzer (Mindray, model BC-6800) using the DIFF channel (using the DIFF reagents provided with Mindray BC-6800), and the white blood cell classification results shown in Table 5 were obtained.

[0565] Table 5 Leukocyte classification results

[0566] As can be seen from Table 5, the white blood cell classification results obtained according to the present invention are substantially consistent with those obtained using the existing BC-6800. This shows that the present invention can simultaneously detect parasites and white blood cells in the blood through a single test of the same blood sample.

[0567] Example 27 Use of the cyanine dye of the present invention in identifying parasites in a blood sample using flow cytometry

[0568] First, prepare dye reagent A8 and hemolytic agent B8 according to the following formula.

[0569] The first dye includes a compound having the above structural formula 4 and is used to dye parasites, and the second dye includes a compound having the following chemical formula and is used to dye cells:

[0570] Then, 20 microliters of dye reagent A8, 1 milliliter of hemolytic agent B8 and 20 microliters of the anticoagulated blood sample of the patient infected with malaria were mixed, and the mixture was incubated at 42°C for 30 seconds to form a sample solution for measurement; then, the sample solution was tested using a flow analyzer with a blue laser with an excitation wavelength of approximately 450 nm to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; a first scatter plot as shown in Figure 42A was generated based on the forward scattered light intensity information and the first fluorescence intensity, and a second scatter plot as shown in Figure 42B was generated based on the side scattered light intensity information and the second fluorescence intensity; based on the first scatter plot shown in Figure 42A, the presence of malarial parasites in the blood sample to be tested could be identified, and based on the second scatter plot shown in Figure 42B, the white blood cells in the blood sample to be tested could be classified into four categories, and the white blood cell classification results shown in Table 6 were obtained.

[0571] The same blood sample was tested on an existing blood analyzer (Mindray, model BC-6800) using the DIFF channel (using the DIFF reagents provided with Mindray BC-6800), and the white blood cell classification results shown in Table 6 were obtained.

[0572] Table 6 Leukocyte classification results

[0573] As can be seen from Table 6, the white blood cell classification results obtained according to the present invention are substantially consistent with those obtained using the existing BC-6800. This shows that the present invention can simultaneously detect parasites and white blood cells in the blood through a single test of the same blood sample.

[0574] The features or combinations of features described in the specification, drawings, and claims may be used in any combination or individually, as long as they are meaningful and not mutually inconsistent within the scope of the present invention. The advantages and features described for the sample analysis method provided by the present invention apply in a corresponding manner to the use of the sample analyzer and DNA-specific dye provided by the present invention, and vice versa.

[0575] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent transformations made by using the contents of the present description and drawings under the inventive concept of the present invention, or direct / indirect application in other related technical fields are included in the patent protection scope of the present invention.

[0576] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound having a structure as shown in general formula I or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, in, R1 and R2 are the same or different and are independently selected from C1-18 straight chain or branched alkyl, C1-18 straight chain or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino; R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C1-6 alkyl, hydroxy, C1-6 alkoxy, and halogenated C1-6 alkyl; Y does not exist or is a counter anion; And limit: When R3 is hydrogen, R1 and R2 are not both methyl and R1 and R2 are not both benzyl.

2. The compound according to claim 1 or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein R1 and R2 are the same or different and are independently selected from C1-6 straight chain alkyl, C1-6 straight chain alkylene-M, where M is selected from sulfonic acid, phenyl, carboxyl, mercapto, and amino.

3. The compound according to claim 1 or 2, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein: At least one of R1 and R2 is a C1-18 linear or branched alkylene-sulfonic acid group.

4. The compound according to any one of claims 1 to 3, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein: R1 and R2 are different and independently selected from C1-6 straight chain alkyl, benzyl, C1-6 straight chain alkylene-carboxyl, C1-6 straight chain alkylene-sulfonic acid, C1-6 straight chain alkylene-mercapto, C1-6 straight chain alkylene-amino.

5. The compound according to any one of claims 1 to 3, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein: R1 and R2 are the same and are selected from C1-6 straight chain alkyl, C1-6 straight chain alkylene-sulfonic acid group, C1-6 straight chain alkylene-carboxyl group.

6. The compound according to any one of claims 1 to 5, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein: R3 is selected from hydrogen, sulfonic acid, halogen, cyano, and C1-6 alkyl.

7. The compound according to any one of claims 1 to 6, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein: When Y is a counter anion, Y is selected from a halide ion (eg, F-, Cl-, Br-, I-), ClO4-, PF6-, CF3SO3-, BF4-, acetate, methanesulfonate, or p-toluenesulfonate.

8. The compound according to any one of claims 1 to 7, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein: When Y is absent, the compound is an internal salt.

9. The compound according to claim 8 or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein At least one of R1 and R2 is selected from a C1-18 linear or branched alkylene-sulfonic acid group and a C1-18 linear or branched alkylene-carboxyl group.

10. The compound according to any one of claims 1 to 9, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein the compound has any of the following structures:

11. The compound according to any one of claims 1 to 9, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein the compound has any of the following structures:

12. A conjugate comprising the compound according to any one of claims 1 to 11 or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof.

13. A composition for staining a biological sample, wherein the composition comprises the compound according to any one of claims 1 to 11 or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, or the conjugate according to claim 12.

14. The composition of claim 13, wherein the biological sample is nucleic acid, preferably deoxyribonucleic acid.

15. Use of the compound of any one of claims 1 to 11, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, the conjugate of claim 12 or the composition of claim 13 for staining a biological sample.

16. The use according to claim 15, wherein the biological sample is nucleic acid, preferably deoxyribonucleic acid.

17. Use of the compound according to any one of claims 1 to 11, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, the conjugate according to claim 12, or the composition according to claim 13, in identifying parasites in a blood sample to be tested using flow cytometry.

18. Use of the compound according to any one of claims 1 to 11 or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, the conjugate according to claim 12 or the composition according to claim 13 in identifying microorganisms in a blood sample or a body fluid sample to be tested by flow cytometry.

19. A sample analysis method, characterized in that: The sample analysis method comprises the following steps: Treating the same biological sample to be tested with a dye reagent containing a first dye and a hemolytic agent for dissolving red blood cells to obtain a sample solution to be tested, wherein the biological sample to be tested is a blood sample to be tested or a body fluid sample to be tested, and the first dye can stain microorganisms; Allowing particles in the sample liquid to pass through an optical detection zone one by one and irradiating the particles passing through the optical detection zone with light, so as to obtain scattered light information and fluorescence information generated by the particles in the sample liquid after being irradiated with light, wherein the fluorescence information includes first fluorescence information from the first dye; and The microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

20. A sample analyzer, characterized in that: The sample analyzer comprises: A sampling device for quantitatively drawing a biological sample to be tested, wherein the biological sample to be tested is a blood sample to be tested or a body fluid sample to be tested; A sample preparation device having a reaction pool and a reagent supply unit, wherein the reaction pool is used to receive the biological sample to be tested sucked by the sampling device, and receive a dye reagent containing a first dye and a hemolytic agent for dissolving red blood cells provided by the reagent supply unit. The biological sample to be tested sucked by the sampling device and the dye reagent and hemolytic agent provided by the reagent supply unit are mixed in the reaction pool to prepare a sample liquid to be tested, wherein the first dye can stain microorganisms; an optical detection device, comprising a light source, a flow cell, a scattered light detector, and a fluorescence detector, wherein the light source is configured to emit a light beam to illuminate the flow cell, the flow cell being in communication with the reaction pool and allowing particles in the sample liquid to pass through the flow cell one by one, the scattered light detector being configured to detect scattered light information generated by particles passing through the flow cell after being illuminated by light, and the fluorescence detector being configured to detect fluorescence information generated by particles passing through the flow cell after being illuminated by light, the fluorescence information including first fluorescence information from the first dye; and The processor is configured to obtain the scattered light information and the fluorescence information from the optical detection device, and identify microorganisms in the sample liquid based on the scattered light information and the first fluorescence information.

21. The sample analyzer according to claim 20, wherein: The processor is further configured to perform the following steps when identifying microorganisms in the sample liquid based on the scattered light information and the first fluorescence information: generating a first scattergram based on the scattered light information, preferably the forward scattered light information, and the first fluorescence information; and Microorganisms in the sample liquid to be tested are identified based on the first scattergram.

22. The sample analyzer according to claim 21, wherein: The processor is further configured to: Acquire a microorganism characteristic region and a white blood cell region from the first scattergram, wherein the intensity of the first fluorescence information of the microorganism characteristic region is greater than the intensity of the first fluorescence information of the white blood cell region; and The microorganisms in the sample liquid to be tested are identified based on the microorganism characteristic region.

23. The sample analyzer according to any one of claims 20 to 22, wherein: The first dye is a dye that can specifically bind to deoxyribonucleic acid, and in particular, the first dye comprises the compound according to any one of claims 1 to 11.

24. A blood analysis method, characterized in that: The blood analysis method comprises the following steps: Treating the same blood sample to be tested with a dye reagent containing a first dye and a hemolytic agent for lysing red blood cells to obtain a sample solution to be tested, wherein the first dye can stain parasites in the blood, and the first dye comprises a compound having a structure of general formula I, in particular comprising the compound according to any one of claims 1 to 11; wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino, R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Y is absent or is a counter anion; Allowing particles in the sample liquid to pass through an optical detection zone one by one and irradiating the particles passing through the optical detection zone with light, so as to obtain scattered light information and fluorescence information generated by the particles in the sample liquid after being irradiated with light, wherein the fluorescence information includes first fluorescence information from the first dye; and The parasites in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

25. A blood analyzer, characterized in that: The blood analyzer comprises: A sampling device, used for quantitatively drawing a blood sample to be tested; A sample preparation device comprising a reaction pool and a reagent supply unit, wherein the reaction pool is used to receive the blood sample to be tested drawn by the sampling device, and to receive a dye reagent containing a first dye and a hemolytic agent for dissolving red blood cells provided by the reagent supply unit. The blood sample to be tested drawn by the sampling device, the dye reagent and the hemolytic agent provided by the reagent supply unit are mixed in the reaction pool to prepare a sample solution to be tested, wherein the first dye comprises a compound having a structure of general formula I, in particular comprising the compound according to any one of claims 1 to 11. wherein R1 and R2 are the same or different and are independently selected from C 1-18 Straight-chain or branched alkyl, C 1-18 Straight or branched alkylene-M, M is selected from sulfonic acid, phenyl, carboxyl, mercapto, amino, R3 is selected from hydrogen, sulfonic acid, halogen, cyano, C 1-6 Alkyl, hydroxyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Y is absent or is a counter anion; an optical detection device, comprising a light source, a flow cell, a scattered light detector, and a fluorescence detector, wherein the light source is configured to emit a light beam to illuminate the flow cell, the flow cell being in communication with the reaction pool and allowing particles in the sample liquid to pass through the flow cell one by one, the scattered light detector being configured to detect scattered light information generated by particles passing through the flow cell after being illuminated by light, and the fluorescence detector being configured to detect fluorescence information generated by particles passing through the flow cell after being illuminated by light, the fluorescence information including first fluorescence information from the first dye; and The processor is configured to obtain the scattered light information and the fluorescence information from the optical detection device, and identify parasites in the sample liquid based on the scattered light information and the first fluorescence information.

26. The blood analyzer according to claim 25, wherein: The processor is further configured to perform the following steps when identifying parasites in the sample liquid based on the scattered light information and the first fluorescence information: generating a first scattergram based on the scattered light information, preferably the forward scattered light information, and the first fluorescence information; and The parasites in the sample liquid to be tested are identified based on the first scatter plot.

27. The blood analyzer according to claim 26, wherein: The processor is further configured to: Acquire a parasite characteristic region and a leukocyte region from the first scattergram, wherein the intensity of the first fluorescence information of the parasite characteristic region is greater than the intensity of the first fluorescence information of the leukocyte region; and The parasites in the sample liquid to be tested are identified based on the parasite characteristic region.

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