WO2024017247A1 - 花菁染料、其制备方法、用途、样本分析方法及分析仪 - Google Patents

花菁染料、其制备方法、用途、样本分析方法及分析仪 Download PDF

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WO2024017247A1
WO2024017247A1 PCT/CN2023/107915 CN2023107915W WO2024017247A1 WO 2024017247 A1 WO2024017247 A1 WO 2024017247A1 CN 2023107915 W CN2023107915 W CN 2023107915W WO 2024017247 A1 WO2024017247 A1 WO 2024017247A1
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Prior art keywords
tested
dye
sample
scattered light
information
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English (en)
French (fr)
Inventor
刘宗俊
陈庚文
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Shenzhen Mindray Bio Medical Electronics Co Ltd
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Shenzhen Mindray Bio Medical Electronics Co Ltd
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Priority claimed from CN202210853485.3A external-priority patent/CN117467734A/zh
Priority claimed from CN202210853495.7A external-priority patent/CN117491319A/zh
Priority claimed from CN202210853531.XA external-priority patent/CN117466834A/zh
Application filed by Shenzhen Mindray Bio Medical Electronics Co Ltd filed Critical Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority to CN202380053930.0A priority Critical patent/CN119522221A/zh
Publication of WO2024017247A1 publication Critical patent/WO2024017247A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/08—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing alicyclic rings
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent materials, e.g. electroluminescent or chemiluminescent
    • C09K11/06—Luminescent materials, e.g. electroluminescent or chemiluminescent containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • the present application relates to a fluorescent dye and its application, in particular to cyanine dye, its preparation method and its use, a sample analysis method and a sample analyzer.
  • the present application also relates to conjugates comprising said cyanine dyes and compositions for staining biological samples.
  • DNA (DeoxyriboNucleic Acid, deoxyribonucleic acid) is a type of biological macromolecule that carries genetic information. Normal cells of organisms generally have relatively stable DNA diploid content, but when pathological changes occur, abnormal changes occur; and the DNA content of different types of organisms generally differs. Therefore, the specific identification and accurate measurement of DNA, especially in living cells, are of great significance.
  • the use of fluorescent dyes for qualitative or quantitative analysis of DNA has aroused the interest of scientific researchers due to its advantages of high sensitivity and fast response.
  • cyanine fluorescent dyes have been widely used as biomolecule fluorescent probes 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 have large molecules, complex structures, and low permeability to living cells. In addition, the Stoke shift of most dyes is small, resulting in severe crosstalk between the excitation spectrum and the emission spectrum, causing background interference and fluorescence self-quenching, which limits their applications.
  • Microorganisms are tiny organisms that exist in nature with small size, simple structure, and invisible to the naked eye. They can only be observed after magnification with the help of special instruments. Microorganisms include prokaryotes (such as bacteria, actinomycetes, mycoplasmas, rickettsiae, chlamydia, and spirochetes), eukaryotes (such as fungi, protozoa, algae), and non-cellular species (viruses and prions).
  • prokaryotes such as bacteria, actinomycetes, mycoplasmas, rickettsiae, chlamydia, and spirochetes
  • eukaryotes such as fungi, protozoa, algae
  • non-cellular species viruses and prions
  • SIRS systemic infection syndrome
  • Immunoassays refer to the detection of specific microorganisms using specific reactions of antigens and antibodies, including traditional agglutinin tests and precipitation tests, as well as new enzyme-linked immunosorbent assays (ELISA), radioactive labeling methods, and chemiluminescence immunoassays. wait. Immunoassays are very specific and faster than traditional culture methods. However, due to shortcomings such as difficulty in obtaining antibodies, high antibody cost, insufficient detection sensitivity, and reactivity easily affected by the environment, immune detection methods are difficult to be widely used in clinical microbial detection.
  • the electrochemical detection method mainly uses the changes in electrical signals generated by the metabolic process of microorganisms to detect microorganisms. Common methods include impedance analysis, potential analysis, current analysis, etc. Equipment based on electrochemical detection methods is small and low-cost, but lacks sensitivity.
  • Blood cell parasitic microorganisms refer to parasites that live in the blood or blood cells of animals, such as malaria or Toxoplasma gondii. Parasites in the blood have a shorter onset period and are more harmful, so early and accurate diagnosis of parasites in blood cells is necessary for effective disease management and monitoring.
  • the first method is the accurate detection and quantification of blood cell-parasitic microorganisms through microscopic examination of blood smears, however this is highly dependent on the training and skills of the operator.
  • the second method is rapid diagnostic testing (RDT) based on antigen-antibody reactions, which does not require high operational skills but is generally expensive and lacks sufficient sensitivity for low-level blood cell parasites.
  • RDT rapid diagnostic testing
  • the third method is to detect parasites in blood cells when using a hematology analyzer for routine blood screening. This method is simple, easy to operate, low-cost and does not rely on the training and skills of the operator.
  • the inventor of the present application obtained a new benzothiazole-based cyanine dye through molecular modification. Its thermal stability is significantly improved, and compared with the existing benzothiazole-based cyanine dye, Dye, the cyanine dye of the present invention has high biological penetration and is more suitable for cell dye and imaging.
  • the application provides a compound having a structure as shown in general formula I or a hydrate, solvate, stereoisomer, tautomer or crystalline form thereof,
  • R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, M is selected from sulfonic acid group, phenyl , carboxyl, thiol, amino;
  • R 3 is selected from hydrogen, sulfonate group, halogen, cyano group, C 1-6 alkyl group, hydroxyl group, C 1-6 alkoxy group, and halogenated C 1-6 alkyl group;
  • Y does not exist or is a counter anion
  • R 3 is hydrogen, R 1 and R 2 are not both methyl and R 1 and R 2 are not both benzyl.
  • R 1 and R 2 may be the same or different.
  • R 1 and R 2 are independently selected from C 1-6 linear alkyl, C 1-6 linear alkylene-M, and M is selected from sulfonate, phenyl, carboxyl, mercapto, Amino.
  • At least one of R 1 and R 2 is a C 1-18 linear or branched alkylene-sulfonate group.
  • R 1 and R 2 are different and independently selected from C 1-6 linear alkyl, benzyl, C 1-6 linear alkylene-carboxy, C 1-6 linear alkylene -Sulfonic acid group, C 1-6 linear alkylene-mercapto group, C 1-6 linear alkylene-amino group.
  • R 1 and R 2 are the same and selected from C 1-6 linear alkyl, C 1-6 linear alkylene-sulfonate, C 1-6 linear alkylene-carboxy.
  • R3 is selected from hydrogen, sulfonate, halogen, cyano, C 1-6 alkyl.
  • Y in general formula I is a counter anion.
  • Y can be selected from halide ions (such as F - , Cl - , Br - , I - ), ClO 4 - , PF 6 - , CF 3 SO 3 - , BF 4 - , acetate, methanesulfonate or p-toluenesulfonate.
  • Y in Formula I is absent, in which case the compound may be an internal salt.
  • Internal salts are also known in the art as “zwitterions”.
  • the compounds of the present invention may contain both an acidic group (such as a sulfonic acid group or a carboxyl group) and a basic group (such as an amino or thiazole ring) within the molecule, the acidic group and the basic group Neutralize each other to form internal salts.
  • each acidic group and/or each basic group may serve as a salt-forming group. Salts formed by various salt formation methods of the compounds are included in the scope of the present invention.
  • At least one of R 1 and R 2 is selected from C 1-18 linear or branched alkylene-sulfonate, C 1-18 linear or branched alkylene-carboxy.
  • the compounds of the present invention may have any of the following structures:
  • a compound of the invention is a compound represented by Structural Formula 5, 6 or 9 above.
  • the compounds of the present invention and their hydrates, solvates, stereoisomers, tautomers or crystal forms can be directly used for staining biological samples, or can also be used in the form of derivatives, and the derivatives include but are not limited to conjugate.
  • Conjugate refers to a compound formed by connecting a compound of the present invention, its hydrate, solvate, stereoisomer, tautomer or crystalline form to other molecules through a covalent bond.
  • Molecules that can be conjugated to the compounds of the invention, their hydrates, solvates, stereoisomers, tautomers or crystalline forms can be molecules that specifically bind to cells or cellular components, including but not limited to antibodies, Antigens, receptors, ligands, enzymes, substrates, coenzymes, etc.
  • the test sample is incubated with a fluorescent conjugate for a period of time so that the fluorescent conjugate specifically binds to certain cells or cellular components in the test sample.
  • the binding of the fluorescent conjugate to cells or cellular components can also be determined by called dyeing.
  • This staining step can be performed multiple times in sequence, or multiple stainings can be performed simultaneously with multiple conjugates.
  • the sample is analyzed in an analytical instrument including an excitation light source that excites the fluorescent dye of the present invention in the conjugate and a measurement device that measures the emitted light generated by the excited fluorescent dye.
  • the present application also provides a composition for staining biological samples, wherein the composition comprises the compound of the present invention, its hydrate, solvate, stereoisomer, tautomer or crystal form, or the compound of the present invention.
  • the biological sample is nucleic acid.
  • the biological sample is DNA.
  • the application also provides the use of the compounds of the invention, their hydrates, solvates, stereoisomers, tautomers or crystal forms, or the conjugates of the invention, or the compositions of the invention in biological samples.
  • the biological sample is nucleic acid.
  • the biological sample is DNA.
  • the application also provides the compounds of the present invention or their hydrates, solvates, stereoisomers, tautomers or crystal forms, or the conjugates of the present invention, or the compositions of the present invention when using flow cytometry. Use of the technique to identify parasites in blood samples to be tested.
  • the parasite is selected from the group consisting of: roundworms, hookworms, tapeworms, Trichomonas vaginalis, liver flukes, Paragonimus westermanii, Toxoplasma gondii, cysticercosis suis, Trichinella spiralis, amoeba , Leishmania donovani, Plasmodium, Schistosoma, filarial worms, hydatid, scabies mites, hair follicle mites, lice, fleas.
  • the application also provides the compounds of the present invention or their hydrates, solvates, stereoisomers, tautomers or crystal forms, or the conjugates of the present invention, or the compositions of the present invention when using flow cytometry.
  • the technology is used to identify microorganisms in blood samples to be tested or body fluid samples to be tested.
  • the microorganism is selected from:
  • Bacteria e.g., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Pertussis pertussis, Diphtheriae, Neisseria meningitidis, Mycobacterium tuberculosis, Clostridium tetani, Leprae, Group A hemolytic chain cocci, Brucella, Bacillus cholerae, Bacillus typhi, Bacillus anthracis, Neisseria gonorrhoeae, Vibrio cholerae, Pseudomonas klebsiella, and Salmonella paratyphi A, B or C),
  • Bacteria e.g., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Pertussis pertussis, Diphtheriae,
  • Viruses such as influenza virus, mumps virus, rubella virus, Japanese encephalitis virus, dengue virus, epidemic hemorrhagic fever virus, rabies virus, human papillomavirus, poliovirus, measles virus, varicella-zoster virus, hepatitis viruses, novel enterovirus 70, coxsackievirus A24 variant, human immunodeficiency virus, poxviruses (e.g. monkeypox virus)),
  • influenza virus mumps virus, rubella virus, Japanese encephalitis virus, dengue virus, epidemic hemorrhagic fever virus, rabies virus, human papillomavirus, poliovirus, measles virus, varicella-zoster virus, hepatitis viruses, novel enterovirus 70, coxsackievirus A24 variant, human immunodeficiency virus, poxviruses (e.g. monkeypox virus))
  • Fungi such as Candida albicans, Trichophyton rubrum, Epidermophyton floccosum
  • Mycoplasma e.g. Mycoplasma pneumoniae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium
  • Chlamydia e.g. Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia domestica
  • Chlamydia e.g. Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia domestica
  • Rickettsiae e.g., Rickettsia prowazekii, Rickettsia morgoni, Rickettsia rickettsiae, Rickettsia scrub typhus
  • Rickettsiae e.g., Rickettsia prowazekii, Rickettsia morgoni, Rickettsia rickettsiae, Rickettsia scrub typhus
  • Actinomycetes e.g. Actinomyces israelensis
  • Spirochetes e.g. Leptospira, Treponema pallidum.
  • blood or body fluid refers to blood or body fluid from a mammal, especially a human.
  • body fluid can be divided into urine, sweat, cerebrospinal fluid, serous cavity fluid, synovial fluid, etc. according to different parts.
  • a small amount of the above-mentioned body fluids exists in the normal human body.
  • the effusion in the serosal cavity includes pleural effusion, ascites and pericardial effusion, and the effusion in the joint cavity is synovial fluid (joint effusion).
  • the compounds of the present invention can be synthesized by general methods in the art.
  • the benzothiazole compounds of the present invention can be synthesized by the following method: first starting from unsubstituted or substituted methylbenzothiazole, and heating it with R 2 X (X is F, Cl, Br or I) The reaction was carried out under reflux to obtain intermediate I in the form of quaternary ammonium salt. Subsequently, the connecting molecule 4-hydroxyisophthalaldehyde and the intermediate I are heated and refluxed (the molar ratio of the intermediate I to 4-hydroxyisophthalaldehyde is greater than or equal to 2), so that the intermediate I and the connecting molecule Condensation gives the benzothiazole compound of the present invention.
  • intermediate I can also be synthesized by the following method: starting from unsubstituted or substituted methylbenzothiazole and other raw materials, and making it with The appropriate sultone undergoes a ring-opening reaction to obtain intermediate I.
  • the benzothiazole compounds of the present invention can be synthesized by the following exemplary method: combining R 1 or R 2 substituted methylbenzothiazole with the connecting molecule 4-hydroxyisophthalaldehyde Heating and refluxing reaction (the molar ratio of 4-hydroxyisophthalaldehyde and methylbenzothiazole substituted by R 1 or R 2 is about 2) to obtain formyl-containing intermediate I'; make the obtained intermediate I' and R 2 or R 1 substituted methylbenzothiazole undergoes a condensation reaction to obtain the benzothiazole compound of the present invention.
  • each intermediate or product can be recovered through separation and purification techniques known in the art to achieve the required purity.
  • C 1-18 linear or branched alkyl refers to a group obtained by removing one hydrogen atom from a linear or branched alkane containing 1 to 18 carbon atoms, specific examples thereof Including but 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, n-octadecyl base.
  • C 1-6 alkyl refers to a group obtained by removing one hydrogen atom from a straight-chain or branched chain alkane containing 1-6 carbon atoms. Specific examples thereof include but are not limited to: base, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, tert-butyl, isobutyl, etc.
  • C 1-6 linear alkyl refers to a group obtained by removing one hydrogen atom from a linear alkane containing 1-6 carbon atoms. Specific examples thereof include but are not limited to: methyl , ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl.
  • C 1-18 straight-chain or branched alkylene refers to a group obtained by removing two hydrogen atoms from a straight-chain or branched alkane containing 1-18 carbon atoms. Specific examples include but are not limited to methylene, Ethylene, propylene, butylene, etc.
  • the term "C 1-6 linear alkylene” refers to a group obtained by removing two hydrogen atoms from a linear alkane containing 1 to 6 carbon atoms.
  • halogen includes fluorine, chlorine, bromine and iodine.
  • halogenated means that a hydrogen on a group or compound is replaced by one or more halogen atoms, including fully halogenated and partially halogenated.
  • C 1-6 alkoxy refers to a group formed in the manner of C 1-6 alkyl-O-.
  • solvate refers to a substance formed by the molecular association of a compound with an organic solvent (eg, methanol, ethanol, propanol, acetonitrile, etc.).
  • organic solvent eg, methanol, ethanol, propanol, acetonitrile, etc.
  • hydrate refers to a substance formed by the association of a compound with water molecules.
  • the term "crystalline form" refers to the crystal structure of a substance. During the crystallization of substances, due to the influence of various factors, the bonding methods within or between molecules change, resulting in different arrangements of molecules or atoms in the crystal lattice space, forming different crystal structures.
  • the compound of the present invention can exist in one crystal structure or in multiple crystal structures, that is, it has "polymorphic form".
  • the compounds of the invention may exist in different crystalline forms.
  • stereoisomer includes conformational isomers and configurational isomers, wherein said conformational isomers
  • Type isomers mainly include cis-trans isomers and optical isomers.
  • the compounds of the present invention may exist in stereoisomeric forms and thus encompass all possible stereoisomeric forms, any combination or any mixture thereof. For example, a single enantiomer, a single diastereomer or a mixture of the above.
  • a compound of the present invention contains an olefinic double bond, it includes both cis and trans isomers, as well as any combination thereof, unless otherwise stated.
  • the compounds described in this invention may exist as tautomeric forms, which have different points of attachment of hydrogens through the displacement of one or more double bonds.
  • a ketone and its enol form are keto-enol tautomers.
  • the present invention encompasses all keto-enol tautomers of the compounds. Each tautomer and mixtures thereof are included within the scope of the invention.
  • the fluorescent dye of the present invention has one or more of the following beneficial effects:
  • Dyes used for cell staining require high cell penetration.
  • the dye of the present invention has high biological penetration and is more suitable for cell dyes and imaging;
  • the dye of the present invention can specifically bind to DNA, which is beneficial to the specific recognition and accurate measurement of DNA;
  • the dye of the present invention has good permeability to living cells, can enter cells to stain nucleic acids without damaging the cell membrane, and has low toxicity and low carcinogenicity;
  • the excitation light of the dye of the present invention is blue-green light with a smaller wavelength, which can identify tiny particles and improve the detection ability of small particles;
  • the dye of the present invention can use ordinary green semiconductor lasers as light sources, which greatly reduces the cost of use;
  • the dye of the present invention has a simple structure, the raw materials for its preparation are easily available, the synthesis yield is high, and it is easy to realize industrialization.
  • another task of the present invention is to provide a sample analysis method and sample analyzer that can accurately, sensitively, cost-effectively and quickly identify based on flow cytometry using fluorescent labeling technology. Microorganisms in blood or body fluids.
  • the second aspect of the present invention proposes a sample analysis method, which includes the following steps:
  • the first dye can dye microorganisms
  • Fluorescence information including first fluorescence information from the first dye
  • Microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.
  • identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes:
  • Microorganisms in the sample fluid to be tested are identified based on the first scatter plot.
  • the scattered light information includes forward scattered light information
  • Generating a first scatter plot based on the scattered light information and the first fluorescence information includes generating the first scatter plot based on the forward scattered light information and the first fluorescence information.
  • identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes:
  • a microbial characteristic area and a leukocyte area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the microbial characteristic area is greater than the intensity of the first fluorescence information of the leukocyte area;
  • Microorganisms in the sample fluid to be tested are identified based on the microorganism characteristic areas.
  • identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes:
  • the number of microorganisms in the sample liquid to be tested is obtained based on the scattered light information and the first fluorescence information.
  • the biological sample to be tested is a blood sample to be tested
  • the sample analysis method further includes:
  • Parasites, especially Plasmodium, in the sample fluid to be tested are identified based on the scattered light information and the first fluorescence information.
  • 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 fluorescence information further includes second fluorescence information from the second dye, wherein, the second dye can stain white blood cells in the blood;
  • the sample analysis method further includes: obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes side scattered light information and forward scattered light information
  • Identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes: identifying microorganisms in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information. ;and
  • Obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information includes: obtaining the leukocyte classification results of the sample liquid to be tested based on the side scattered light information and the second fluorescence information. Classification results.
  • 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 fluorescence information further includes second fluorescence information from the second dye
  • the second dye can stain white blood cells and nucleated red blood cells in the blood
  • the sample analysis method further includes: obtaining at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes forward scattered light information
  • Identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes: identifying microorganisms in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information. ;and
  • Obtaining at least one of the white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information includes: based on the forward scattered light information and The second fluorescence information obtains at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample liquid to be tested.
  • illuminating the particles flowing through the optical detection zone with light includes illuminating the particles flowing through the optical detection zone with light of a single wavelength.
  • the first dye is a dye that specifically binds to DNA.
  • the first dye includes a compound having the structure of Formula I above, wherein R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, M is selected from sulfonate group, phenyl, carboxyl, mercapto, amino group, R 3 is selected from hydrogen, sulfonate group, halogen, cyano group, C 1-6 Alkyl group, hydroxyl group, C 1-6 alkoxy group, halogenated C 1-6 alkyl group, Y does not exist or is a counter anion.
  • R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, M is selected from sulfonate group, phenyl, carboxyl, mercapto, amino group, R 3 is selected from hydrogen, sulfonate group, halogen, cyano group
  • a third aspect of the present invention proposes a sample analyzer, including:
  • a sampling device used to quantitatively absorb biological samples to be tested, where the biological samples to be tested are blood samples to be tested or body fluid samples to be tested;
  • a sample preparation device having a reaction tank and a reagent supply part, wherein the reaction tank is used to receive the biological sample to be tested drawn by the sampling device, and to receive a dye containing a first dye provided by the reagent supply part Reagents and hemolytic reagents for lysing red blood cells.
  • the biological sample to be tested absorbed by the sampling device is mixed with the dye reagent and hemolytic reagent provided by the reagent supply part in the reaction tank to prepare a sample to be tested.
  • Liquid, wherein the first dye can dye microorganisms
  • Optical detection device including 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 cell and the sample liquid to be tested is The particles in the flow chamber can pass through the flow chamber one by one, the scattered light detector is used to detect the scattered light information generated by the particles passing through the flow chamber after being irradiated by light, and the fluorescence detector is used to detect the particles passing through the flow chamber. Fluorescence information generated by the particles after being illuminated by light, the fluorescence information including first fluorescence information from the first dye; and
  • a processor configured to acquire the scattered light information and the fluorescence information from the optical detection device, and identify microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information.
  • the processor is further configured to perform the following steps when identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information:
  • Microorganisms in the sample fluid to be tested are identified based on the first scatter plot.
  • the scattered light information includes forward scattered light information
  • the processor is further configured to generate the first scatter plot based on the forward scattered light information and the first fluorescence information.
  • the processor is further configured to:
  • a microbial characteristic area and a leukocyte area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the microbial characteristic area is greater than the intensity of the first fluorescence information of the leukocyte area;
  • Microorganisms in the sample fluid to be tested are identified based on the microorganism characteristic areas.
  • the processor is further configured to perform the following steps when identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information:
  • the number of microorganisms in the sample liquid to be tested is obtained based on the scattered light information and the first fluorescence information.
  • the biological sample to be tested is a blood sample to be tested
  • the processor is further configured to:
  • Parasites, especially Plasmodium, in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.
  • 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 second dye can stain white blood cells in the blood, and the The fluorescence information also includes second fluorescence information from the second dye;
  • the processor is further configured to: obtain a leukocyte classification result of the sample fluid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes side scattered light information and forward scattered light information
  • the processor is further configured to: identify the test target based on the forward scattered light information and the first fluorescence information. microorganisms in the sample liquid, and obtain the leukocyte classification result of the sample liquid to be tested based on the side scattered light information and the second fluorescence information.
  • the biological sample to be tested is a blood sample to be tested
  • the dye reagent further includes a second dye that is different from the first dye, and the second dye can detect white blood cells and nucleated red blood cells in the blood. staining, the fluorescence information further comprising second fluorescence information from a second dye;
  • 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 fluid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes forward scattered light information
  • the processor is further configured to: identify microorganisms in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information, and identify microorganisms in the sample liquid to be tested based on the forward scattered light information and the second fluorescence information.
  • the information obtains at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested.
  • the light source is configured to illuminate the flow chamber with a single wavelength of light.
  • the first dye is a dye that specifically binds to DNA.
  • the first dye includes a compound having the structure of Formula I above, wherein R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, M is selected from sulfonate group, phenyl, carboxyl, mercapto, amino group, R 3 is selected from hydrogen, sulfonate group, halogen, cyano group, C 1-6 Alkyl group, hydroxyl group, C 1-6 alkoxy group, halogenated C 1-6 alkyl group, Y does not exist or is a counter anion.
  • R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, M is selected from sulfonate group, phenyl, carboxyl, mercapto, amino group, R 3 is selected from hydrogen, sulfonate group, halogen, cyano group
  • a dye capable of staining microorganisms and a hemolytic agent used to dissolve red blood cells are used to process the blood sample to be tested or the body fluid sample to be tested, To obtain the sample liquid to be tested, and then use flow cytometry to obtain the scattered light information and fluorescence information of the sample liquid to be tested. Based on the scattered light information and the fluorescence information, it can be identified whether there are microorganisms in the sample liquid to be tested. This enables accurate, sensitive, low-cost and rapid detection of microorganisms in blood or body fluids.
  • another task of the present invention is to provide a blood analysis method and a blood analyzer that can perform low-cost routine blood testing based on flow cytometry and using fluorescent labeling technology. Simple, sensitive, fast and accurate detection of parasites in blood.
  • the fourth aspect of the present invention provides a blood analysis method, including the following steps:
  • the first dye includes a compound having the structure of the above general formula I, wherein R 1 and R 2 are the same or different, and are independently selected from C 1-18 straight chain or branched alkyl, C 1-18 straight chain or Branched alkylene-M, M is selected from sulfonate group, phenyl, carboxyl, mercapto, amino group, R 3 is selected from hydrogen, sulfonate group, halogen, cyano group, C 1-6 alkyl group, hydroxyl group, C 1 -6 alkoxy group, halogenated C 1-6 alkyl group, Y does not exist or is a counter anion;
  • Fluorescence information including first fluorescence information from the first dye
  • Parasites in the sample fluid to be tested are identified based on the scattered light information and the first fluorescence information.
  • identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes:
  • Parasites in the sample fluid to be tested are identified based on the first scatter plot.
  • the scattered light information includes forward scattered light information
  • Generating a first scatter plot based on the scattered light information and the first fluorescence information includes generating the first scatter plot based on the forward scattered light information and the first fluorescence information.
  • identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes:
  • a parasite characteristic area and a leukocyte area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the parasite characteristic area is greater than the intensity of the first fluorescence information of the leukocyte area;
  • Parasites in the sample fluid to be tested are identified based on the parasite characteristic area.
  • identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes:
  • the number of parasites in the sample liquid to be tested is obtained based on the scattered light information and the first fluorescence information.
  • the blood analysis method further includes:
  • Microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.
  • the compound according to the first aspect of the present invention can be used as the compound included in the first dye.
  • the compound according to the first aspect of the present invention can be used as the compound included in the first dye.
  • the dye reagent further includes a second dye different from the first dye, the second dye is capable of staining white blood cells and nucleated red blood cells in the blood, and the fluorescence information further includes information from the second dye the second fluorescence information;
  • the blood analysis method further includes: obtaining at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes forward scattered light information
  • Identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes: identifying parasites in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information. parasites; and
  • Obtaining at least one of the white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information includes: based on the forward scattered light information and The second fluorescence information obtains at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample liquid to be tested.
  • the dye reagent further includes a second dye that is different from the first dye, the second dye is capable of staining white blood cells in the blood, and the fluorescence information further includes a second fluorescence from the second dye. information;
  • the blood analysis method further includes: obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes side scattered light information and forward scattered light information
  • Identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes: identifying parasites in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information. parasites; and
  • Obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information includes: obtaining the leukocyte classification results of the sample liquid to be tested based on the side scattered light information and the second fluorescence information. Classification results.
  • illuminating the particles flowing through the optical detection zone with light includes illuminating the particles flowing through the optical detection zone with light of a single wavelength.
  • a fifth aspect of the present invention provides a blood analyzer, including:
  • a sample preparation device having a reaction tank and a reagent supply part, wherein the reaction tank is used to receive the blood sample to be tested drawn by the sampling device, and to receive a dye containing a first dye provided by the reagent supply part Reagents and hemolytic reagents for lysing red blood cells.
  • the blood sample to be tested drawn by the sampling device is mixed with the dye reagent and hemolytic reagent provided by the reagent supply part in the reaction tank to prepare a sample to be tested.
  • the first dye includes a compound with the structure of the above general formula I, wherein R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1 -18 Linear or branched alkylene-M, M is selected from sulfonate group, phenyl, carboxyl, mercapto, amino group, R 3 is selected from hydrogen, sulfonate group, halogen, cyano group, C 1-6 alkyl group , hydroxyl group, C 1-6 alkoxy group, halogenated C 1-6 alkyl group, Y does not exist or is a counter anion;
  • Optical detection device including 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 cell and the sample liquid to be tested is The particles in the flow chamber can pass through the flow chamber one by one, the scattered light detector is used to detect the scattered light information generated by the particles passing through the flow chamber after being irradiated by light, and the fluorescence detector is used to detect the particles passing through the flow chamber. Fluorescence information generated by the particles after being illuminated by light, the fluorescence information including first fluorescence information from the first dye; and
  • a processor configured to acquire the scattered light information and the fluorescence information from the optical detection device, and identify parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information.
  • the processor is further configured to perform the following steps when identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information:
  • Parasites in the sample fluid to be tested are identified based on the first scatter plot.
  • the scattered light information includes forward scattered light information
  • the processor is further configured to generate the first scatter plot based on the forward scattered light information and the first fluorescence information.
  • the processor is further configured to:
  • a parasite characteristic area and a leukocyte area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the parasite characteristic area is greater than the intensity of the first fluorescence information of the leukocyte area;
  • Parasites in the sample fluid to be tested are identified based on the parasite characteristic area.
  • the processor is further configured to perform the following steps when identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information:
  • the number of parasites in the sample liquid to be tested is obtained based on the scattered light information and the first fluorescence information.
  • the processor is further configured to:
  • Microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.
  • the compound according to the first aspect of the present invention can be used as the compound included in the first dye.
  • the compound according to the first aspect of the present invention can be used as the compound included in the first dye.
  • the dye reagent further includes a second dye different from the first dye, the second dye is capable of staining white blood cells and nucleated red blood cells in the blood, and the fluorescence information further includes information from the second dye the second fluorescence information;
  • 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 fluid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes forward scattered light information
  • the processor is further configured to: identify parasites in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information, and identify parasites in the sample liquid to be tested based on the forward scattered light information and the second fluorescence information.
  • the fluorescence information obtains at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested.
  • the dye reagent further includes a second dye that is different from the first dye, the second dye is capable of staining white blood cells in the blood, and the fluorescence information further includes a second fluorescence from the second dye. information;
  • the processor is further configured to: obtain a leukocyte classification result of the sample fluid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes side scattered light information and forward scattered light information
  • the processor is further configured to: identify parasites in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information, and identify parasites in the sample liquid to be tested based on the side scattered light information and the second fluorescence information.
  • the fluorescence information is used to obtain the leukocyte classification result of the sample liquid to be tested.
  • the light source is configured to illuminate the flow chamber with a single wavelength of light.
  • a cyanine dye capable of staining parasites especially a benzothiazole-based cyanine dye and a cyanine dye used to dissolve red blood cells are used.
  • the blood sample to be tested is treated with a hemolytic agent to obtain the sample liquid to be tested, and then flow cytometry is used to obtain the scattered light information and fluorescence information of the sample liquid to be tested. Based on the scattered light information and the fluorescence information, the sample to be tested can be identified. Test the sample fluid for the presence of parasites. This enables accurate, sensitive, low-cost and rapid detection of parasites in blood.
  • Figure 1 is a schematic flow chart of an embodiment of a sample analysis method for identifying microorganisms according to the present invention.
  • Figure 2 is a first scatter plot when the biological sample to be tested is a blood sample according to an embodiment of the present invention.
  • Figure 3 is a first scatter plot when the biological sample to be tested is a body fluid sample according to an embodiment of the present invention.
  • Figure 4 is a schematic flow chart of another embodiment of a sample analysis method for identifying microorganisms according to the present invention.
  • Figure 5 is a scatter plot of microbial identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to the present invention.
  • Figure 6 is a schematic flow chart of yet another embodiment of a sample analysis method for identifying microorganisms according to the present invention.
  • Figure 7 is a scatter plot of microbial identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to the present invention.
  • Figure 8 is a schematic diagram of the emission spectra of two dyes according to an embodiment of the present invention.
  • Figure 9 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.
  • Figure 10 is a schematic flow chart of an embodiment of a blood analysis method for identifying parasites according to the present invention.
  • Figure 11 is a first scatter plot for identifying parasites according to an embodiment of the present invention.
  • Figure 12 is a schematic flow chart of another embodiment of a blood analysis method for identifying parasites according to the present invention.
  • Figure 13 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to the present invention
  • Figure 14 is a schematic flow chart of yet another embodiment of a blood analysis method for identifying parasites according to the present invention.
  • Figure 15 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to the present invention.
  • Figure 16 is a schematic structural diagram of an embodiment of a sample analyzer according to the present invention.
  • 17 to 19 are structural schematic diagrams of different embodiments of the optical detection device according to the present invention.
  • Figure 20 shows the changes in fluorescence spectrum of the dye in Example 7 as the DNA concentration increases.
  • Figure 21 shows the changes in fluorescence spectrum of the dye in Example 7 as the RNA concentration increases.
  • Figure 22 is a linear relationship diagram between fluorescence intensity and calf thymus DNA and RNA concentration in Example 7.
  • Figure 23 shows the results of observing the staining of HepG2 living cells by Compound II under a laser microscope in Example 8.
  • Figure 24 shows the results of observing the staining of HepG2 living cells by Compound III under a laser microscope in Example 9.
  • Figure 25 shows the evaluation results of dye stability in Example 10.
  • Figure 26 shows the evaluation results of the cell penetration of the dye in Example 11.
  • Figure 27 is a scatter plot of microbial identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 12 of the present invention.
  • Figure 28 is a scatter plot obtained by testing body fluid samples according to Embodiment 13 of the present invention.
  • Figure 29 is a scatter plot of microbial identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 14 of the present invention.
  • Figure 30 is a scatter plot obtained by testing body fluid samples according to Embodiment 15 of the present invention.
  • Figure 31 is a scatter plot of microbial identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 16 of the present invention.
  • Figure 32 is a scatter plot obtained by testing body fluid samples according to Embodiment 17 of the present invention.
  • Figure 33 is a scatter plot of microbial identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to Embodiment 18 of the present invention.
  • Figure 34 is a scatter plot obtained by testing body fluid samples according to Embodiment 19 of the present invention.
  • Figure 35 is a scatter plot of microbial identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to Embodiment 20 of the present invention.
  • Figure 36 is a scatter plot obtained by testing body fluid samples according to Embodiment 21 of the present invention.
  • Figure 37 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to Embodiment 22 of the present invention.
  • Figure 38 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to Embodiment 23 of the present invention.
  • Figure 39 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to Embodiment 24 of the present invention.
  • Figure 40 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 25 of the present invention.
  • Figure 41 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 26 of the present invention.
  • Figure 42 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 27 of the present invention.
  • first ⁇ second ⁇ third involved in the embodiment of the present invention are only used to distinguish similar objects and do not represent a specific ordering of objects. It is understandable that “first ⁇ second ⁇ third” Three” may interchange specific order or precedence where permitted.
  • blood cell analyzers also known as blood analyzers and hemocytometers
  • flow cytometry have the advantages of rapid, accurate and extremely simple operation for detecting blood cells. It is an indispensable fully automated rapid blood cell detection instrument in modern clinical testing.
  • the present invention proposes a technical solution for rapid detection of pathogenic microorganisms in blood or body fluids based on flow cytometry and fluorescent labeling technology.
  • microorganisms refer to tiny organisms that are difficult to see clearly with the naked eye and require an optical microscope or an electron microscope to be observed. Microorganisms include bacteria, viruses, fungi and a few algae.
  • an embodiment of the present invention first provides a sample analysis method 100 for rapid detection of 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.
  • the same biological sample to be tested is processed using a dye reagent containing the first dye and a hemolytic agent for lysing red blood cells to obtain a sample liquid to be tested.
  • the biological sample to be tested is a blood sample to be tested or a body fluid sample to be tested.
  • the first dye can stain the microorganisms. That is to say, when there are microorganisms in the blood sample to be tested or the body fluid sample to be tested, using the first dye to process the blood sample to be tested or the body fluid sample to be tested can cause the microorganisms therein to be stained.
  • the hemolyzing agent and the dye reagent can be added to the same blood sample or body fluid sample to be tested successively to obtain the sample liquid to be tested, and then the sample liquid to be tested is incubated so that the dye reagent can be fully To stain the substance to be tested in the sample liquid to be tested.
  • the dye reagent may be mixed with the hemolyzing agent in advance 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 250:1-1000:1. After mixing evenly, the mixture The obtained sample liquid to be tested is incubated at a temperature of 25°C to 50°C for 10 seconds to 1 minute, preferably 20 seconds to 40 seconds.
  • the hemolytic agent is used to dissolve red blood cells in blood or body fluids and split the red blood cells into fragments, but can keep the morphology of white blood cells basically unchanged.
  • the hemolytic agent may include any one or a combination of cationic surfactants, nonionic surfactants, anionic surfactants, amphiphilic surfactants, and buffer pairs.
  • the cationic surfactant is, for example, selected from at least one or a combination of dodecyltrimethylammonium chloride, octyltrimethylammonium bromide, and tetradecyltrimethylammonium chloride.
  • the nonionic surfactant is, for example, at least one or a combination of several selected from the group consisting of long-chain fatty alcohol polyoxyethylene, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ether, and fatty amine polyoxyethylene ether.
  • the buffer pair is, for example, selected from at least one or a combination of several types of phosphates, citrates, and Tris-HCl.
  • the anionic surfactant is, for example, selected from the group consisting of dodecylbenzene sulfonic acid, sodium fatty alcoholyl sulfate, sodium ethoxylated fatty acid methyl ester sulfonate, sodium secondary alkyl sulfonate, alcohol ether carboxylate at least one or a combination of several.
  • the hemolytic agent may include at least one of alkyl glycosides, triterpene saponins, and steroidal saponins.
  • the particles in the sample liquid to be tested are allowed to pass through the optical detection area one by one and the particles flowing through the optical detection area are irradiated with light to obtain the scattered light information and fluorescence generated by the particles in the sample liquid to be tested after being irradiated with light.
  • the fluorescence information at least includes the first fluorescence information from the first dye. That is to say, the first fluorescence information includes the fluorescence signal generated under light excitation after the particles in the sample liquid to be tested are combined with the first dye.
  • the scattered light information and fluorescence information of the sample liquid to be tested are obtained based on the principle of flow cytometry.
  • light such as a laser beam
  • the characteristics of the particles themselves such as volume, staining degree, cell content size and content, cell nucleus density, etc.
  • the scattered light at various angles corresponding to the characteristics can be received by the signal detector to obtain light information related to the structure and composition of the particles, that is, the scattered light information and fluorescence information of the present invention.
  • the scattered light information includes, for example, 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 reflects the complexity of the internal structure of the cell (such as intracellular particles or nuclei)
  • fluorescence reflects the nucleic acid in the cell. substance content. This light information can be used to identify various types of particles in the sample liquid to be tested.
  • the scattered light information includes scattered light signal intensity
  • the fluorescence information includes fluorescence signal intensity
  • step S130 microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.
  • step S130 that is, identifying microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information may include: generating a first scatter plot based on the scattered light information and the first fluorescence information; and based on the first Scatter plots identify microorganisms in the sample fluid to be tested.
  • the scatter plot may be a two-dimensional scatter plot or a three-dimensional scatter plot, on which two-dimensional or three-dimensional feature information of multiple particles is distributed, where the X coordinate axis and Y coordinate of the scatter plot Both the axes and the Z coordinate axis represent a characteristic of each particle.
  • the X-axis represents the forward scattered light signal intensity
  • the Y-axis represents the fluorescence signal intensity
  • the Z-axis represents the side-scattered light signal intensity.
  • the scatter plots in this article are not limited to graphical form, and can also be in data form, such as numerical forms of tables or lists with the same or similar resolution as the scatter plots, or in any form that has been used in this field. Know of other suitable ways to present it.
  • the scattered light information may include forward scattered light information FSC, especially forward scattered light signal intensity.
  • generating the first scattergram based on the scattered light information and the first fluorescence information may include: based on the forward scattered light information FSC, especially the forward scattered light signal intensity and the first fluorescence information FL1, especially the first fluorescence signal.
  • a first scatter plot of intensity is generated, as shown in Figures 2 and 3. Wherein, FIG. 2 shows the first scatter plot when the biological sample to be tested is a blood sample, and FIG. 3 shows the first scatter plot when the biological sample to be tested is a body fluid sample.
  • identifying microorganisms in the sample liquid to be tested based on scattered light information and first fluorescence information may include:
  • the microbial characteristic area P1 and the white blood cell area P2 are obtained from the first scatter plot, and the intensity of the first fluorescence information of the microbial characteristic area is greater than the intensity of the first fluorescence information of the white blood cell area;
  • Microorganisms in the sample liquid to be tested are identified based on the microbial characteristic area P1.
  • the forward scattered light information is the abscissa and the first fluorescence information is the ordinate, such as the two-dimensional scatter plot shown in Figures 2 and 3, in 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 of the white blood cell area) Signal intensity), when there are microorganisms in the blood sample or body fluid sample, there are always clusters of scattered points in this characteristic area, and when there are no microorganisms in the blood sample or body fluid sample, there are no clusters of scattered points in this characteristic area.
  • the scattered points of the group in a scatter plot in which the forward scattered light information is the abscissa and the first fluorescence information is the ordinate, such as the two-dimensional scatter plot shown in Figures 2 and 3, in There is a specific area above the white blood cell group (i.e., the white blood cell area) (i.e., the first fluor
  • clusters of scattered points in this characteristic area can be used to characterize microbial particle clusters, and this characteristic area can also be called a microbial characteristic area.
  • this characteristic area can also be called a microbial characteristic area.
  • identifying microorganisms in the sample fluid to be tested based on scattered light information and first fluorescence information may include: obtaining microorganisms in the sample fluid to be tested based on scattered light information, especially forward scattered light information and first fluorescence information.
  • Number of microorganisms For example, in the embodiments shown in Figures 2 and 3, the number of scattered points in the microbial characteristic area P1 can be used to characterize the number of microorganisms in the sample liquid to be tested.
  • scatter point data of a large number of biological samples containing microorganisms can be collected in advance, and the microorganisms can be obtained through fitting
  • the correlation curve between the scatter point data and the actual quantity is obtained to obtain the corresponding calculation model, such as a linear calculation model.
  • the microbial scatter point data of the biological sample to be tested is obtained according to the method of the present invention. Based on the microbial scatter point data of the biological sample to be tested and the above-mentioned predetermined calculation model, the biological sample to be tested can be estimated. The number of microorganisms in the microorganisms, thereby achieving quantitative analysis of microorganisms.
  • the sample analysis method 100 may further include: identifying the sample liquid to be tested based on the scattered light information and the first fluorescence information.
  • the parasites in the sample liquid to be tested are particularly identified based on the first scatter plot. This enables simultaneous detection of microorganisms and parasites in blood through a single test of the same biological sample to be tested, improving blood detection efficiency and saving reagent costs without increasing blood consumption.
  • the dye reagent may further include a second dye different from the first dye, and the second dye can affect the blood. cells are stained.
  • the fluorescence information obtained in step S120 also includes the second fluorescence information FL2 from the second dye, especially the second fluorescence signal intensity. That is, the second fluorescence information includes the particles in the sample liquid to be tested and the second dye. The fluorescence signal generated under light excitation after binding.
  • the cell parameters of the sample fluid to be tested such as white blood cell parameters, nucleated red blood cell parameters, etc., can be further obtained based on the scattered light information and the second fluorescence information.
  • a second dye capable of staining white blood cells in the blood is used in step S110.
  • the sample analysis method 100 may also include step S140: obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.
  • the white blood cells in the sample fluid to be tested can be classified into a lymphocyte group, a monocyte group, a neutrophil group, and an eosinophil group based on the scattered light information and the second fluorescence information, and the Various types of white blood cells are counted to obtain lymphocyte count and/or the ratio of lymphocyte count to white blood cell count, monocyte count and/or monocyte count to the ratio of white blood cell count, neutrophil count and/or neutrophil count granulocyte count as a ratio of white blood cell count, eosinophil count, and/or eosinophil count as a ratio of white blood cell count.
  • the white blood cells in the sample fluid to be tested can also be classified into a lymphocyte population, a monocyte population, a neutrophil population, an eosinophil population, and a basophil population based on the scattered light information and the second fluorescence information. granulocyte population, and count various types of white blood cells. This enables simultaneous detection of microorganisms and white blood cells in the blood through one test of the same blood sample to be tested, improving blood detection efficiency and saving reagent costs without increasing blood consumption.
  • the second dye used for leukocyte classification can be a nucleic acid dye that can bind to nucleic acid substances in cells, for example, including cyanine cationic compounds.
  • a nucleic acid dye that can bind to nucleic acid substances in cells, for example, including cyanine cationic compounds.
  • the second dye used for leukocyte classification is a non-DNA or RNA-specific nucleic acid dye, including, for example, a compound of Formula II below.
  • the scattered light information may include side scattered light information SSC and forward scattered light information FSC.
  • identifying the microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information 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.
  • step S140 may include: obtaining the sample to be tested based on the side scattered light information SSC and the second fluorescence information FL2
  • the leukocyte classification results of the liquid are shown in Figure 5B.
  • a second scatter plot is generated based on the side scattered light information and the second fluorescence information.
  • the white blood cells in the sample fluid to be tested are divided into neutrophils and lymphocytes based on gating technology. population, monocyte population, and eosinophil population and count these cell populations.
  • step S140 may also include obtaining the leukocyte 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.
  • a second dye capable of staining nucleated cells in the blood is used in step S110.
  • the second dye can be used in particular to identify nucleated red blood cells.
  • the sample analysis method 100 may also include step S150: 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. At least one, especially based on the scattered light information and the second fluorescence information, obtains the white blood cell count, the basophil count and the nucleated red blood cell count of the sample fluid to be tested. This enables simultaneous detection of microorganisms in blood and detection of white blood cells and/or nucleated red blood cells through one test of the same blood sample to be tested, improving blood detection efficiency and saving reagent costs without increasing blood consumption.
  • the second dye used for staining nucleated cells can also be a nucleic acid dye that can bind to nucleic acid substances in the cell.
  • the second dye used for staining nucleated cells includes, for example, a compound having the following chemical formula III.
  • the scattered light information may include forward scattered light information FSC.
  • identifying microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information may include: based on the forward scattered light information FSC and the first fluorescence information FL1 to identify the microorganisms in the sample liquid to be tested, as shown in Figure 7A; and based on the scattered light information and the second fluorescence information, the white blood cell count, basophil count and active granulocyte count of the sample liquid to be tested are obtained.
  • At least one of the nucleated red blood cell count may include: obtaining at least one of the white blood cell count, the basophil count, and the nucleated red blood cell count of the sample fluid to be tested based on the forward scattered light information FSC and the second fluorescence information FL2. A, as shown in Figure 7B.
  • irradiating the particles flowing through the optical detection zone with light includes: using a single wavelength of Light, in particular blue light, for example with a wavelength of around 450 nanometers, illuminates the particles flowing through the optical detection zone.
  • the first dye and the second dye are selected so that the peaks of the emission spectra of the first dye and the second dye correspond
  • the absolute value of the wavelength difference is greater than 30 nanometers and less than 80 nanometers.
  • the first dye and the second dye are selected such that the emission spectra of the first dye and the second dye overlap by no more than 50%.
  • Figure 8 shows a schematic diagram of the emission spectra of the first dye and the second dye.
  • the curve represented by the dotted line is the emission spectrum 210 of the first dye
  • the curve represented by the solid line is the emission spectrum 220 of the second dye.
  • the peak point of the emission spectrum 210 of the first dye is A
  • the peak point of the emission spectrum 220 of the second dye is D.
  • the absolute difference between the abscissas of peak point A and peak point D ie, the difference in wavelengths corresponding to the peaks
  • the overlap amount of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye may be a ratio of the area of the first polygon to the area of the second polygon, where the area of the first polygon is equal to point E, The area of the curved polygon surrounded by point G and point C, and the area of the second polygon is equal to the curve formed by the emission spectrum 210 of the first dye (or the emission spectrum 220 of the second dye) and the baseline 230
  • the area of the side polygon, where the reference line 230 is a dotted horizontal line parallel to the horizontal axis as shown in Figure 8.
  • the dotted horizontal line is at the normalized peak of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye. 5%.
  • Points E and F are respectively the left and right intersection points of the emission spectrum 220 of the second dye and the reference line 230.
  • Points B and point C are respectively the left and right intersection points of the emission spectrum 210 of the first dye and the reference line 230.
  • the overlap of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye is no more than 50%.
  • the absolute value of the difference in 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, more preferably greater than 50 nanometers and less than 70 nanometers, so as to further reduce the detection interference between the first fluorescence signal and the second fluorescence signal.
  • the overlap of the emission spectra of the first dye and the second dye is no more than 35%, preferably no more than 15%, thereby further reducing the detection interference between the first fluorescence signal and the second fluorescence signal.
  • the smaller the overlap of the emission spectra of the first dye and the second dye the more conducive it is to distinguish the first fluorescence signal and the second fluorescence signal.
  • the first dye can be a large Stokes shift dye.
  • a large Stokes shift dye refers to a dye whose emission spectrum and excitation spectrum have wavelength differences corresponding to respective peaks greater than a predetermined threshold.
  • Figure 9 is a schematic spectrum diagram of a large Stokes shift dye.
  • the excitation spectrum (also called absorption spectrum) 310 of the large Stokes shift dye is shown by the dotted line, and the emission spectrum 320 is shown by the solid line.
  • the excitation spectrum is 310
  • the peak point of is A1
  • the peak point of emission spectrum 320 is A2.
  • the difference between the abscissas of the peak point A2 and the peak point A1 ie, the difference in wavelengths corresponding to the respective peaks of the emission spectrum and the excitation spectrum
  • the predetermined threshold may be, for example, greater than 30 nanometers and less than 150 nanometers, preferably greater than 50 nanometers and less than 100 nanometers.
  • the use of dyes with a large Stokes shift can in particular reduce mutual detection interference of the first fluorescent signal and the second fluorescent signal.
  • the first dye is a dye that can specifically bind to deoxyribonucleic acid (ie, DNA), such as a cyanine dye, especially a benzothiazole-based cyanine dye.
  • the first dye may include a compound having the structure of Formula I:
  • R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, and M is selected from sulfonic acid group, Phenyl, carboxyl, mercapto, amino; R 3 is selected from hydrogen, sulfonic acid group, halogen, cyano, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy, halogenated C 1-6 alkyl; And Y is absent or is a counter anion.
  • the first dye including a compound with the structure of general formula I proposed by the present invention has one or more of the following advantages: good thermal stability; high biological (microorganism, cell) penetrating power; capable of specificity It combines well with DNA, which is beneficial to the specific recognition and accurate measurement of DNA; it has good permeability to living cells and can enter cells to stain nucleic acids without damaging the cell membrane. It has low toxicity and low carcinogenicity; it can be used It can be excited by blue-green light with a smaller wavelength, so that it can identify tiny particles and improve the detection ability of small particles; it can use ordinary green semiconductor lasers as light sources, which greatly reduces the cost of use; it has a simple structure and the raw materials for its preparation are easy to obtain. The synthesis yield is high and easy to realize industrialization.
  • R 1 and R 2 are independently selected from C 1-6 linear alkyl, C 1-6 linear alkylene-M, and M is selected from sulfonate, phenyl, carboxyl, mercapto, Amino.
  • At least one of R 1 and R 2 is a C 1-18 linear or branched alkylene-sulfonic acid group, a C 1-18 linear or branched alkylene-carboxy group.
  • R 1 and R 2 are different and independently selected from C 1-6 linear alkyl, benzyl, C 1-6 linear alkylene-carboxy, C 1-6 linear alkylene -Sulfonic acid group, C 1-6 linear alkylene-mercapto group, C 1-6 linear alkylene-amino group. In other embodiments, R 1 and R 2 are the same and selected from C 1-6 linear alkyl, C 1-6 linear alkylene-sulfonate, C 1-6 linear alkylene-carboxy .
  • R3 is selected from hydrogen, sulfonate, halogen, cyano, C 1-6 alkyl.
  • R 3 when R 3 is hydrogen, R 1 and R 2 are not simultaneously methyl and R 1 and R 2 are not simultaneously benzyl.
  • Y in Formula I is a counter anion.
  • Y can be selected from halide ions (such as F - , Cl - , Br - , I - ), ClO 4 - , PF 6 - , CF 3 SO 3 - , BF 4 - , acetate, methanesulfonate or p-toluenesulfonate.
  • Y in Formula I is absent, in which case the compound may be an internal salt.
  • “Internal salts” are also known in the art as "zwitterions”.
  • the compounds of the present invention may contain acidic groups (such as sulfonic acid groups or carboxyl group) and a basic group (such as an amino group or a thiazole ring), the acidic group and the basic group neutralize each other to form an internal salt.
  • acidic groups such as sulfonic acid groups or carboxyl group
  • basic group such as an amino group or a thiazole ring
  • each acidic group and/or each basic group may serve as a salt-forming group. Salts formed by various salt formation methods of the compounds are included in the scope of the present invention.
  • the compound of the present invention having the structure of Formula I may have any structure shown in the following table:
  • the compound of the present invention is a compound represented by the above structural formula 5, 6 or 9.
  • R 1 and R 2 are each independently selected from C 1-18 alkyl, C 1-18 sulfonic acid group, C 1-18 carboxyl, C 1-18 hydroxyl, The group consisting of C 1-18 NR 5 R 6 , benzyl and substituted benzyl, wherein the substituent of the substituted benzyl is selected from C 1-18 alkyl, CN, COOH, NH 2 , NO 2 , OH, The group consisting of SH, C 1-6 alkoxy, C 1-6 alkylamino, C 1-6 amido, halogen and C 1-6 haloalkyl, preferably R 1 and R 2 are the same C 1-18 sulfonate Acid group; R 3 is selected from the group consisting of H, C 1-18 sulfonic acid group, phenyl, OR 6 and halogen; and Y- is a negative ion.
  • the dye reagent of the present invention can be stored in a water-soluble organic phase such as glycerol, glycol, and ethylene glycol.
  • the dye reagent of the present invention can be stored alone or mixed with a hemolytic agent.
  • the present invention also proposes a technical solution for rapid detection of parasites in blood based on flow cytometry and fluorescent labeling technology.
  • the parasites are selected from the group consisting of: roundworms, hookworms, tapeworms, Trichomonas vaginalis, liver flukes, Paragonimus westermani, Toxoplasma gondii, cysticercosis suis, Trichinella spiralis, amoeba, Leishmania donovani, Plasmodium, schistosomiasis, filarial worms, hydatid, scabies mites, hair follicle mites, lice, fleas.
  • an embodiment of the present invention also 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.
  • step S1100 the same blood sample to be tested is processed using a dye reagent containing the first dye and a hemolytic agent for lysing red blood cells to obtain a sample liquid to be tested.
  • the first dye can stain parasites in the blood. That is to say, when there are parasites in the blood sample to be tested, using the first dye to process the blood sample to be tested can cause the parasites in the blood sample to be tested to be stained.
  • the first dye is a cyanine dye, especially a cyanine dye of the benzothiazole type and includes compounds having the structure of the general formula I:
  • R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, and M is selected from sulfonic acid group, Phenyl, carboxyl, mercapto, amino, R 3 is selected from hydrogen, sulfonic acid group, halogen, cyano, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy, halogenated C 1-6 alkyl, Y is absent or is a counter anion.
  • a hemolyzing agent and a dye reagent can be added to the same blood sample to be tested successively to obtain a sample liquid to be tested, and then the sample liquid to be tested is incubated so that the dye reagent can fully stain the sample to be tested.
  • the substance to be measured in the liquid may also be mixed with the hemolytic agent in advance to obtain a mixed mixture.
  • step S110 of the method 100 can be correspondingly applied to the hemolytic agent used in step S1100, and will not be described again.
  • step S110 of the method 100 can be correspondingly applied to the compound of the first dye used in step S1100, and will not be described again.
  • the particles in the sample liquid to be tested are allowed to pass through the optical detection area one by one and the particles flowing through the optical detection area are irradiated with light to obtain the scattered light information and fluorescence generated by the particles in the sample liquid to be tested after being irradiated with light.
  • the fluorescence information at least includes the first fluorescence information from the first dye. That is to say, the first fluorescence information includes the fluorescence signal generated under light excitation after the particles in the sample liquid to be tested are combined with the first dye.
  • the scattered light information and fluorescence information of the sample liquid to be tested are obtained based on the principle of flow cytometry.
  • light such as a laser beam
  • the characteristics of the particles themselves such as volume, staining degree, cell content size and content, cell nucleus density, etc.
  • the scattered light at various angles corresponding to the characteristics can be received by the signal detector to obtain light information related to the structure and composition of the particles, that is, the scattered light information and fluorescence information of the present invention.
  • the scattered light information includes, for example, 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 reflects the complexity of the internal structure of the cell (such as intracellular particles or nuclei)
  • fluorescence reflects the nucleic acid in the cell. substance content. This light information can be used to identify various types of particles in the sample liquid to be tested.
  • the scattered light information includes scattered light signal intensity
  • the fluorescence information includes fluorescence signal intensity
  • step S1300 parasites in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.
  • step S1300 that is, identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information, may include: generating a first scatter plot based on the scattered light information and the first fluorescence information; and based on the first scattered light information and the first fluorescence information.
  • a scatter plot identifies parasites in the sample fluid being tested.
  • the scattered light information may include forward scattered light information FSC, especially forward scattered light signal intensity.
  • generating the first scattergram based on the scattered light information and the first fluorescence information may include: based on the forward scattered light information FSC, especially the forward scattered light signal intensity and the first fluorescence information FL1, especially the first fluorescence signal.
  • a first scatter plot of intensity is generated, as shown in Figure 11.
  • identifying parasites in the sample liquid to be tested based on scattered light information and first fluorescence information may include:
  • the parasite characteristic area P1 and the white blood cell area P2 are obtained from the first scatter plot, and the intensity of the first fluorescence information of the parasite characteristic area is greater than the intensity of the first fluorescence information of the white blood cell area;
  • Parasites in the sample liquid to be tested are identified based on the parasite characteristic area P1.
  • clusters of scattered points in this characteristic area can be used to characterize parasite particle clusters, and this characteristic area can also be called a parasite characteristic area.
  • this characteristic area can also be called a parasite characteristic area.
  • identifying parasites in the sample liquid to be tested based on scattered light information and first fluorescence information may include: obtaining parasites in the sample liquid to be tested based on scattered light information, especially forward scattered light information and first fluorescence information. number of parasites. For example, in the embodiment shown in FIG. 11 , the number of scattered points in the parasite characteristic area P1 can be used to characterize the number of parasites in the sample liquid to be tested.
  • scatter point data of a large number of parasite-containing blood samples (such as the number of scatter points in the parasite characteristic area P1) and the actual number of parasites in these blood samples can be collected in advance, and the data can be simulated by The correlation curve between the scatter point data of the parasite and the actual number is obtained by combining it, thereby obtaining the corresponding calculation model, such as a linear calculation model.
  • the parasite scatter point data of the blood sample to be tested is obtained according to the method of the present invention. Based on the parasite scatter point data of the blood sample to be tested and the above-mentioned predetermined calculation model, the parasite scatter point data of the blood sample to be tested can be estimated. The number of parasites in the blood sample, enabling quantitative analysis of parasites.
  • the blood analysis method 1000 may further include: identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information, especially based on the first scatter plot. Identify microorganisms in the sample fluid to be tested. This enables simultaneous detection of microorganisms and parasites in the blood through one test of the same blood sample to be tested, improving blood detection efficiency and saving reagent costs without increasing blood consumption.
  • the dye reagent of the present invention can be stored in a water-soluble organic phase such as glycerol, glycol, and ethylene glycol.
  • the dye reagent of the present invention can be stored alone or mixed with a hemolytic agent.
  • the dye reagent may further include a second dye different from the first dye, and the second dye can stain cells in the blood.
  • the fluorescence information obtained in step S1200 also includes the second fluorescence information FL2 from the second dye, especially the second fluorescence signal intensity. That is, the second fluorescence information includes the particles in the sample liquid to be tested and the second dye. The fluorescence signal generated under light excitation after binding.
  • the cell parameters of the sample fluid to be tested such as white blood cell parameters, nucleated red blood cell parameters, etc., can be further obtained based on the scattered light information and the second fluorescence information.
  • a second dye that can stain nucleated cells in the blood is used in step S1100.
  • the second dye can be used in particular to identify nucleated red blood cells.
  • the blood analysis method 1000 may also include step S1400: 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. At least one, especially based on the scattered light information and the second fluorescence information, obtains the white blood cell count, the basophil count and the nucleated red blood cell count of the sample fluid to be tested.
  • the second dye used to stain nucleated cells may be a nucleic acid dye that can bind to nucleic acid substances in the cell, or a protein dye that can bind to protein substances in the cell.
  • the second dye used for staining nucleated cells includes, for example, a compound having the following chemical formula III.
  • the scattered light information may include forward scattered light information FSC.
  • identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information 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.
  • step S1400 may include: At least one of the white blood cell count, the basophil count, and the nucleated red blood cell count of the sample fluid to be tested is obtained based on the forward scattered light information FSC and the second fluorescence information FL2, as shown in FIG. 13B .
  • a second dye capable of staining white blood cells in the blood is used in step S1100.
  • the blood analysis method 1000 may also include step S1500: obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.
  • the white blood cells in the sample fluid to be tested can be classified into a lymphocyte group, a monocyte group, a neutrophil group, and an eosinophil group based on the scattered light information and the second fluorescence information, and the Various types of white blood cells are counted to obtain lymphocyte count and/or the ratio of lymphocyte count to white blood cell count, monocyte count and/or monocyte count to the ratio of white blood cell count, neutrophil count and/or neutrophil count granulocyte count as a ratio of white blood cell count, eosinophil count, and/or eosinophil count as a ratio of white blood cell count.
  • the white blood cells in the sample fluid to be tested can also be classified into a lymphocyte population, a monocyte population, a neutrophil population, an eosinophil population, and a basophil population based on the scattered light information and the second fluorescence information. granulocyte population, and count various types of white blood cells. As a result, it is possible to simultaneously detect parasites and leukocytes in the blood through one test of the same blood sample to be tested, improving blood detection efficiency and saving reagent costs without increasing blood consumption.
  • the second dye used for leukocyte classification can also be a nucleic acid dye that can bind to nucleic acid substances in cells, for example, including cyanine cationic compounds.
  • a nucleic acid dye that can bind to nucleic acid substances in cells, for example, including cyanine cationic compounds.
  • the second dye used for leukocyte classification is a non-DNA or RNA-specific nucleic acid dye, including, for example, a compound of Formula II below.
  • the scattered light information may include side scattered light information SSC and forward scattered light information FSC.
  • identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information may include: Identify 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 Figure 15A; and obtain the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information, that is, Step S1500 may include: obtaining the white blood cell classification result of the sample liquid to be tested based on the side scattered light information SSC and the second fluorescence information FL2, such as the four white blood cell classification results as described above, as shown in Figure 15B.
  • a second scatter plot is generated based on the side scattered light information and the second fluorescence information.
  • the white blood cells in the sample fluid to be tested are divided into neutrophils and lymphocytes based on gating technology. population, monocyte population, and eosinophil population and count these cell populations.
  • step S1500 may also include obtaining the leukocyte 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.
  • irradiating the particles flowing through the optical detection zone with light includes: using a single wavelength of Light, in particular blue light, for example with a wavelength of around 4500 nanometers, illuminates the particles flowing through the optical detection zone.
  • the first dye and the second dye are selected so that the peaks of the emission spectra of the first dye and the second dye are The absolute value of the corresponding wavelength difference is greater than 30 nanometers and less than 80 nanometers.
  • the first dye and the second dye are selected such that the emission spectra of the first dye and the second dye overlap by no more than 50%, see Figure 8 .
  • the absolute value of the difference in 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, more preferably greater than 50 nanometers and less than 70 nanometers, so as to further reduce the detection interference between the first fluorescence signal and the second fluorescence signal.
  • the overlap of the emission spectra of the first dye and the second dye is no more than 35%, preferably no more than 15%, thereby further reducing the detection interference between the first fluorescence signal and the second fluorescence signal.
  • the smaller the overlap of the emission spectra of the first dye and the second dye the more conducive it is to distinguish the first fluorescence signal and the second fluorescence signal.
  • the first dye can be a large Stokes shift dye.
  • a large Stokes shift dye refers to a dye whose emission spectrum and excitation spectrum have wavelength differences corresponding to respective peaks greater than a predetermined threshold. Referring again to FIG. 9 , the difference between the abscissas of the peak point A2 and the peak point A1 is greater than the predetermined threshold.
  • the predetermined threshold may be, for example, greater than 30 nanometers and less than 150 nanometers, preferably greater than 50 nanometers and less than 100 nanometers.
  • the use of dyes with a large Stokes shift can in particular reduce mutual detection interference of the first fluorescent signal and the second fluorescent signal.
  • the present invention also provides a sample analyzer 400 based on flow cytometry and fluorescent labeling technology.
  • the sample analyzer 400 includes a sampling device 410 , a sample preparation device 420 , an optical detection device 430 and a processor 440 .
  • the sample analyzer 400 also includes a liquid circuit system (not shown) for communicating with the sampling device 410, the sample preparation device 420, and the optical detection device 430 to facilitate liquid transmission between these devices.
  • the sampling device 410 is used to quantitatively absorb biological samples to be tested, where the biological samples to be tested are blood samples to be tested or body fluid samples to be tested.
  • the sampling device 410 has a pipette with a pipette nozzle and a driving device, which is used to drive the pipette to quantitatively suck the biological sample to be tested through the pipette nozzle.
  • the sampling device can collect the collected organisms to be tested
  • the sample is transported to sample preparation device 420.
  • the sample preparation device 420 has a reaction cell and a reagent supply part.
  • the reaction pool is used to receive the biological sample to be tested sucked by the sampling device 410, and to receive the dye reagent containing the first dye and the hemolytic agent for dissolving red blood cells provided by the reagent supply unit.
  • the biological sample to be tested sucked by the sampling device 410 and The dye reagent and hemolytic reagent provided by the reagent supply department are mixed in the reaction tank to prepare the sample liquid to be tested.
  • the first dye can dye microorganisms.
  • the first dye is capable of staining parasites and includes a compound having the structure of general formula I:
  • R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, and M is selected from sulfonic acid group, Phenyl, carboxyl, mercapto, amino, R 3 is selected from hydrogen, sulfonic acid group, halogen, cyano, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy, halogenated C 1-6 alkyl, Y is absent or is a counter anion.
  • further examples of the first dye may refer to the above various embodiments of the first dye used in the sample analysis method 100 and the various embodiments of the first dye used in the blood analysis method 1000. The embodiments will not be described again here.
  • 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 cell and the particles in the sample liquid to be measured can pass through the flow chamber one by one, scattering
  • the light detector is used to detect the scattered light information generated by the particles passing through the flow chamber after being illuminated by light
  • the fluorescence detector is used to detect the fluorescence information generated by the particles passing through the flow chamber after being illuminated by light.
  • the fluorescence information includes the first dye from the first dye. a fluorescence information.
  • a flow cell refers to a chamber of a focused fluid flow suitable for detecting light scattering signals and fluorescence signals.
  • a particle such as a blood cell
  • the particle scatters the incident light beam from the light source directed to the detection hole in all directions.
  • Photodetectors may be positioned at one or more different angles relative to the incident light beam to detect light scattered by the particles to obtain a light scattering signal. Since different particles have different light scattering properties, the light scattering signal can be used to distinguish different particle populations.
  • the light scattering signal detected near the incident light beam is often referred to as the forward light scattering signal or the small angle light scattering signal.
  • the forward light scatter signal can be detected from an angle of about 1° to about 10° from the incident light beam. In other embodiments, the forward light scatter signal may be detected from an angle of about 2° to about 6° from the incident light beam.
  • Light scattering signals detected at approximately 90° to the incident beam are often referred to as side light scattering signals. In some embodiments, the side light scatter signal may be detected from an angle of about 65° to about 115° with respect to the incident light beam.
  • fluorescent signals from blood cells stained with fluorescent dyes are also detected at approximately 90° to the incident light beam.
  • 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.
  • Optical detection device 430 preferably includes forward scattered light detection detector and side scattered light detector.
  • FIG. 17 shows a specific example of the optical detection device 430.
  • the optical detection device 430 has a light source 401, a beam shaping component 402, a flow chamber 403 and a forward scattered light detector 404 arranged in sequence on a straight line.
  • a dichroic mirror 406 is arranged at an angle of 45° to the straight line.
  • Part of the lateral light emitted by the particles in the flow chamber 403 passes through the dichroic mirror 406 and is captured by the fluorescence detector 405 arranged behind the dichroic mirror 106 at an angle of 45° to the dichroic mirror 406; the other part
  • the side light is reflected by the dichroic mirror 406 and captured by a side scattered light detector 407 arranged in front of the dichroic mirror 406 at an angle of 45°.
  • the processor 440 is used to process the optical signals collected by the optical detection device 430 to obtain the required results. For example, a two-dimensional scatter plot or a three-dimensional scatter plot can be generated based on various collected optical signals, and the scatter plot can be generated in the scatter plot. In the figure, particle analysis is performed based on the gating method.
  • the processor 440 can also perform visual processing on the intermediate operation result or the final operation result, and then display it through the display device 450 .
  • the processor 440 includes, but is not limited to, a central processing unit (Central Processing Unit, CPU), a micro control unit (Micro Controller Unit, MCU), a field-programmable gate array (Field-Programmable Gate Array, FPGA), a digital A device such as a signal processor (DSP) used to interpret computer instructions and process data in computer software.
  • the processor 440 is used to execute each computer application program in a computer-readable storage medium, so that the sample analyzer 400 executes a corresponding detection process and analyzes the optical signal detected by the optical detection device 430 in real time.
  • the sample analyzer 400 also includes a first housing 460 and a second housing 470 .
  • the display device 450 may be, for example, a user interface.
  • the optical detection device 130 and the processor 140 are disposed inside the second housing 170 .
  • the sample preparation device 420 is, for example, disposed inside the first casing 460
  • the display device 450 is, for example, disposed on the outer surface of the first casing 460 and is used to display the detection results of the blood cell analyzer.
  • a computer having a display may be remotely communicatively connected to the sample analyzer 400, such as being installed remotely from the laboratory where the blood cell analyzer is located, such as in a doctor's office.
  • 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 to be tested based on the scattered light information and the first fluorescence information.
  • the processor 440 may be further configured to perform the following steps when identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information:
  • Microorganisms in the sample liquid to be tested are identified based on the first scatter plot.
  • the scattered light information includes forward scattered light information
  • the processor 440 may be further configured to generate a first scatter plot based on the forward scattered light information and the first fluorescence information.
  • the processor 440 may also be configured to:
  • the microbial characteristic area and the white blood cell area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the microbial characteristic area is greater than the intensity of the first fluorescence information of the white blood cell area;
  • the processor 440 may be further configured to perform the following steps when identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information: based on the scattered light information and the first fluorescence information. The information obtains the number of microorganisms in the sample liquid to be tested.
  • the processor 440 may be further configured to: identify parasites, especially Plasmodium, in the sample fluid to be tested based on the scattered light information and the first fluorescence information.
  • the biological sample to be tested is a blood sample to be tested
  • the dye reagent may further include a second dye different from the first dye, and the second dye can stain cells in the blood.
  • the fluorescence information obtained by the optical detection device 430 also includes second fluorescence information from the second dye.
  • the cell parameters of the sample fluid to be tested such as white blood cell parameters, nucleated red blood cell parameters, etc., can be further obtained based on the scattered light information and the second fluorescence information.
  • FIG. 18 shows another specific example of the optical detection device 430.
  • the optical detection device 430 has a laser 431, a front light assembly 432, a flow chamber 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 used to detect the first fluorescence signal corresponding to the first dye generated by the particles passing through the flow chamber 433 after being irradiated with light
  • the second fluorescence detector 439 is used to detect when the particles passing through the flow chamber 433 are irradiated by light.
  • a second fluorescent signal corresponding to the second dye is generated after light irradiation.
  • the laser 431, the front light assembly 432, the flow chamber 433 and the forward scattered light detector 434 are sequentially arranged on the optical axis along the optical axis direction, and the front light assembly is configured to cause the excitation light emitted by the laser 431 to flow in the particles.
  • the particles converge in the detection area of the flow chamber 433 in the direction, so that the particles flowing through the detection area of the flow chamber 433 can generate scattered light.
  • a first dichroic mirror 435 is arranged at an angle of 45° to the optical axis.
  • a part of the side light generated by the particles when flowing through the detection area of the flow chamber 433 is reflected by the first dichroic mirror 435 and captured by the side scattered light detector 436, while the other part of the side light passes through the first dichroic mirror 435.
  • the dichroic mirror 435 reaches a second dichroic mirror 437 , which is also arranged downstream of the first dichroic mirror 435 at an angle of 45° to the optical axis.
  • a part of the lateral light that passes through the first dichroic mirror 435 is reflected by the second dichroic mirror 437 and captured by the first fluorescence detector 438, while the other part passes through the second dichroic mirror 437 and is captured by the second fluorescence detector.
  • Detector 439 captures.
  • the forward scattered light detector 434 may also be arranged tilted to the optical axis.
  • a reflective mirror 4341 is arranged downstream of the flow chamber along the optical axis direction, which reflects the forward scattered light of the particles into a forward scattered light detector 434 arranged obliquely to the optical axis.
  • the second dye is a dye capable of staining leukocytes in the blood, in particular a dye used for leukocyte classification.
  • the processor 440 may also be configured to: obtain the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes side scattered light information and forward scattered light information.
  • the processor 440 may 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 the microorganisms in the sample liquid to be tested based on the side scattered light information and the second fluorescence information. WBC differential results.
  • the second dye is a dye capable of staining leukocytes and nucleated red blood cells in the blood, in particular a dye for identifying nucleated red blood cells.
  • the processor 440 may be further configured to: obtain at least one of the white blood cell count, the basophil count, and the nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes forward scattered light information.
  • the processor 440 may 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 the microorganisms in the sample liquid to be tested based on the forward scattered light information and the second fluorescence information. At least one of a white blood cell count, a basophil count, and a nucleated red blood cell count.
  • the light source may be configured to illuminate the flow chamber with a single wavelength of light.
  • the optical detection device 430 only has one laser source that emits blue-green light, especially a laser source that emits blue light.
  • sample analyzer 400 for more embodiments and advantages of the sample analyzer 400 according to the first embodiment of the present invention, please refer to the above description of the sample analysis method 100 of the present invention, and will not be described again here.
  • 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 to be tested based on the scattered light information and the first fluorescence information.
  • the processor 440 may be further configured to perform the following steps when identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information:
  • Parasites in the sample liquid to be tested are identified based on the first scatter plot.
  • the scattered light information includes forward scattered light information
  • the processor 440 may be further configured to generate a first scatter plot based on the forward scattered light information and the first fluorescence information.
  • the processor 440 may also be configured to:
  • the parasite characteristic area and the white blood cell area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the parasite characteristic area is greater than the intensity of the first fluorescence information of the white blood cell area;
  • the processor 440 may be further configured to perform the following steps when identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information: based on the scattered light information and the first fluorescence information. Fluorescence information is used to obtain the number of parasites in the sample liquid to be tested.
  • the processor 440 may be further configured to: identify microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information.
  • the dye agent may further comprise a second dye different from the first dye, the second dye being capable of staining cells in the blood.
  • the fluorescence information obtained by the optical detection device 430 also includes second fluorescence information from the second dye.
  • the cell parameters of the sample fluid to be tested such as white blood cell parameters, nucleated red blood cell parameters, etc., can be further obtained based on the scattered light information and the second fluorescence information.
  • the second dye is a dye capable of staining white blood cells and nucleated red blood cells in the blood, in particular a dye used to identify nucleated red blood cells.
  • the processor 440 may be further configured to: obtain at least one of the white blood cell count, the basophil count, and the nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes forward scattered light information.
  • the processor 440 may also be 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 the sample liquid to be tested based on the forward scattered light information and the second fluorescence information. At least one of a white blood cell count, a basophil count, and a nucleated red blood cell count.
  • the second dye is a dye capable of staining leukocytes in the blood, in particular a dye used for leukocyte classification.
  • the processor 440 may also be configured to: obtain the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.
  • the scattered light information includes side scattered light information and forward scattered light information.
  • the processor 440 may also be 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 the sample liquid to be tested based on the side scattered light information and the second fluorescence information. WBC classification results.
  • the light source may be configured to illuminate the flow chamber with a single wavelength of light.
  • the optical detection device 430 only has one laser source that emits blue-green light, especially a laser source that emits blue light.
  • the present invention also proposes that DNA-specific dyes (that is, dyes that can specifically bind to deoxyribonucleic acid) are used in flow cytometry. Cytometry is used to identify microorganisms in blood samples to be tested.
  • DNA-specific dyes include compounds having the structure of Formula I:
  • R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, and M is selected from sulfonic acid group, Phenyl, carboxyl, mercapto, amino; R 3 is selected from hydrogen, sulfonic acid group, halogen, cyano, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy, halogenated C 1-6 alkyl; And Y is absent or is a counter anion.
  • the present invention also proposes the application of DNA-specific dyes (ie dyes that can specifically bind to deoxyribonucleic acid) in identifying parasites in blood samples to be tested using flow cytometry.
  • DNA-specific dyes include compounds with the structure of general formula I:
  • R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, and M is selected from sulfonic acid group, Phenyl, carboxyl, mercapto, amino; R 3 is selected from hydrogen, sulfonic acid group, halogen, cyano, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy, halogenated C 1-6 alkyl; And Y is absent or is a counter anion.
  • the structure of compound I can be represented by structural formula 1.
  • compound (E)-4-2-(5-formyl-2-hydroxystyryl)benzothiazole-3-butyl-1-sulfonic acid inner salt (structure See the right side of reaction equation I).
  • reaction scheme I The yield of reaction scheme I is approximately 33%.
  • the second step is to prepare 4-(2-((E)-2-((E)-3-((Z)))-2-(3-methylbenzothiazole-2(3H)) according to the following reaction formula II -Alkenyl)vinyl)-6-oxo-1,4-cyclohexadien-1-yl)ethyl)benzothiazol-3-yl)butanesulfonate inner salt (Compound I).
  • the structure of compound II can be represented by structural formula 2.
  • 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).
  • compound II (4-(2-((E)-6-oxo-3-((Z))-2-(3-(4-sulfobutyl))benzo [d]thiazol-2-ylidene)ethylene)cyclohexan-1,4-dien-1-yl)vinyl)benzo[d]thiazol-3-yl)butanesulfonic acid.
  • the structure of compound VI can be represented by structural formula 6, where Y- is an iodide ion.
  • 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 white powder solid.
  • the white powdery solid is 2,3-dimethyl-5-chlorobenzothiazole quaternary ammonium iodide (right side of reaction formula I).
  • the yield of Scheme I is approximately 67%.
  • reaction mixture is filtered, and the filter cake is collected to obtain a crude product.
  • the above 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 about 0.46 mmol of brown-black solid.
  • the brown-black solid powder was 5-chloro-2-((E)-2-((E)-3-((Z)-2-(5-) Chloro-3-methylbenzothiazole-2(3H)-ylidene)vinyl)-6-oxocyclohexan-1,4-dien-1-yl)vinyl-3-methylbenzothiazole Salt (Compound VI), the yield of reaction formula II is about 70%.
  • the structure of compound VII can be represented by structural formula 7.
  • 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).
  • reaction formula I was about 78%.
  • compound VIII can be represented by structural formula 8, where Y- is an iodide ion.
  • 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).
  • 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 7.1 mmol of white powder solid B.
  • the white powdery solid is 2,3-dimethyl-5-fluorobenzothiazole quaternary ammonium iodide (the structure is shown on the right side of Reaction Formula I).
  • the yield of Scheme I is approximately 79%.
  • the reaction mixture is filtered, and the filter cake is washed three times with 10 mL acetonitrile.
  • the solid is collected to obtain about 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-methylbenzothiazole-2(3H)-ylidene)vinyl)-6-oxocyclohexan-1, 4-Dien-1-yl)vinyl-3-methylbenzothiazolium salt (compound VIII), the yield of reaction formula II is about 95%.
  • the structure of compound IX can be represented by structural formula 9, where Y- is an iodide ion.
  • 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).
  • 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).
  • 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 2.18 mmol of yellow powder solid, which was 2,3-dimethyl-5-cyanobenzothiazole quaternary ammonium iodide (the structure is shown on the right side of Reaction Formula II).
  • the yield of reaction scheme II is approximately 38%.
  • the reaction mixture was filtered, and the filter cake was washed with acetonitrile, and the filter cake was collected to obtain a crude product.
  • the brown black solid powder was 2-((trans)-2-((trans)-6-oxo-3-((Z )-2-(5-cyanobenzothiazol-2(3H)-yl)vinyl)cyclohexan-1,4-dienyl)vinyl-3-(5-cyano)benzothiazolium salt ( Compound IX), the yield of reaction formula III is about 52%.
  • calf thymus DNA Prepares an aqueous solution of calf thymus DNA with a certain concentration, measure its absorbance value at 260nm with a UV absorption spectrophotometer, and calibrate its concentration to 1.8mM. Take 100 ⁇ L of calf thymus DNA that has been calibrated to 1.8 mM, and add 290 ⁇ L of water to dilute the calf thymus DNA aqueous solution to 0.5 mM. Prepare a DMSO (dimethyl sulfoxide) solution of Compound B with a concentration of 1mM, take 1.5 ⁇ L, add M-60LN hemolytic reagent (Mindray) to 3mL, place it in a cuvette, and measure its fluorescence intensity.
  • DMSO dimethyl sulfoxide
  • Figure 20 shows the changes in the fluorescence spectrum of compound II as the DNA concentration increases.
  • Figure 21 shows the changes in the fluorescence spectrum of compound II as the RNA concentration increases.
  • Figure 22 is a linear relationship between fluorescence intensity and calf thymus DNA and RNA concentration.
  • 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.
  • Figure 23 shows the observations.
  • the middle picture is a white field micrograph of Compound II staining HepG2 living cells.
  • the left picture is a fluorescence micrograph of Compound II staining HepG2 living cells.
  • the right picture is a superposition of the bright field image and the fluorescence image. As shown in the figure, it can be observed that compound II clearly stains the nucleus of HepG2 cells, indicating that the dye has good permeability and strong nucleic acid staining ability.
  • Figure 24 shows the observations.
  • the middle picture is a white field micrograph of Compound III staining HepG2 living cells.
  • the left picture is a fluorescence micrograph of Compound III staining HepG2 living cells.
  • the right picture is a superposition of the bright field image and the fluorescence image. As shown in the figure, it can be observed that compound III stains HepG2 cell nuclei clearly, indicating that the dye has good permeability and strong nucleic acid staining ability.
  • Dyes used in commercial cell dyes must have certain high-temperature stability. Existing dyes have poor high-temperature stability and are subject to certain limitations in actual commercial applications. In order to improve this situation, the present invention designs and develops a variety of new dyes from the perspective of molecular structure in order to improve dye stability.
  • Figure 25 shows the degradation curve.
  • the four curves from top to bottom correspond to dye 5, dye 6, dye 9, and dye QCy-DT.
  • the degradation of dye QCy-DT is the most serious, while the degradation of dye 5, dye 6, and dye 9 is milder, indicating that the introduction of an electron-withdrawing group at compound R3 can improve the stability of the compound to a certain extent. , which is beneficial to commercial applications such as blood cell staining of compounds.
  • Dyes used in commercial cell dyes need to have good cell penetration. Due to the small logP value of the molecular structure of existing dyes (the logarithmic value of the ratio of the distribution coefficient of the compound in n-octanol and water), the penetration of living cells is difficult. The permeability is weak and is subject to certain limitations in actual commercial applications. In order to improve this situation, the present invention is designed from the perspective of molecular structure and introduces a variety of chemical groups to improve the stability of the dye and at the same time enhance the penetrating power of the dye molecules into cells.
  • Figure 26 shows the test results.
  • the four curves correspond to dye 6, dye 9, dye 5, and dye QCy-DT from top to bottom.
  • dyes 5, 6, and 9 have better cell penetration than dye QCy-DT.
  • Example 12 Use of the cyanine dye of the present invention in identifying microorganisms in blood samples to be tested using flow cytometry
  • the first dye includes a compound with the above structural formula 1 and is used for staining microorganisms
  • the second dye includes a compound with the following chemical formula and is used for staining cells:
  • the same blood sample to be tested was tested using the DIFF channel (using the DIFF supporting reagent of Mindray BC-6800) on the existing blood analyzer (Mindray, model BC-6800), and the leukocyte classification shown in Table 1 was obtained. result.
  • the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously achieve microbial detection and leukocyte detection in blood through one detection of the same blood sample to be tested.
  • Example 13 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples to be tested using flow cytometry
  • a certain amount of Pseudomonas Klebsiella was added to physiological saline to obtain a simulated body fluid sample to be tested; the above simulated body fluid sample to be tested was tested using the reagents and methods of Example 12 to obtain forward The scattered light intensity FSC and the first fluorescence intensity FL1; the first scatter plot shown in Figure 28 is generated based on the forward scattered light intensity information and the first fluorescence intensity; it can be identified based on the first scatter plot shown in Figure 28 The presence of microorganisms in simulated body fluid samples.
  • Example 14 Use of the cyanine dye of the present invention in identifying microorganisms in blood samples to be tested using flow cytometry
  • the first dye includes a compound with the above-mentioned structural formula 2 and is used for staining microorganisms
  • the second dye includes a compound with the following chemical formula and is used for staining cells:
  • the same blood sample to be tested was tested using the DIFF channel (using Mindray BC-6800's DIFF supporting reagent) on the existing blood analyzer (Mindray, model BC-6800), and the leukocyte classification shown in Table 2 was obtained. result.
  • the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously achieve microbial detection and leukocyte detection in blood through one detection of the same blood sample to be tested.
  • Example 15 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples to be tested using flow cytometry
  • a certain amount of Pseudomonas Klebsiella was added to the physiological saline to obtain a simulated body fluid sample to be tested; the above simulated body fluid sample to be tested was tested using the reagents and methods of Example 14 to obtain forward The scattered light intensity FSC and the first fluorescence intensity FL1; the first scatter plot shown in Figure 30 is generated based on the forward scattered light intensity information and the first fluorescence intensity; it can be identified based on the first scatter plot shown in Figure 11 The presence of microorganisms in simulated body fluid samples.
  • Example 16 Use of the cyanine dye of the present invention in identifying microorganisms in blood samples to be tested using flow cytometry
  • the first dye includes a compound with the above-mentioned structural formula 3 and is used for staining microorganisms
  • the second dye includes a compound with the following chemical formula and is used for staining cells:
  • the sample liquid to be tested is tested; then, a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm is used to test the sample liquid to be tested to obtain the forward scattered light intensity FSC, side scattered light intensity SSC, and the first Fluorescence intensity FL1 and second fluorescence intensity FL2; generate a first scatter plot as shown in Figure 31A based on the forward scattered light intensity information and the first fluorescence intensity, and generate a first scatter plot as shown in Figure 31A based on the side scattered light intensity information and the second fluorescence intensity A second scatter plot as shown in FIG. 31B is generated; based on the first scatter plot as shown in FIG.
  • the same blood sample to be tested was tested using the DIFF channel (using Mindray BC-6800's DIFF supporting reagent) on the existing blood analyzer (Mindray, model BC-6800), and the leukocyte classification shown in Table 3 was obtained. result.
  • the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously achieve microbial detection and leukocyte detection in blood through one detection of the same blood sample to be tested.
  • Example 17 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples to be tested using flow cytometry
  • a certain amount of Escherichia coli is added to the physiological saline to obtain a simulated body fluid sample to be tested; the above 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 third 1.
  • Fluorescence intensity FL1 generate a first scatter diagram as shown in Figure 32 based on the forward scattered light intensity information and the first fluorescence intensity; based on the first scatter diagram shown in Figure 32, it can be identified that in the simulated body fluid sample Microorganisms are present.
  • Example 18 Use of the cyanine dye of the present invention in identifying microorganisms in blood samples to be tested using flow cytometry
  • the first dye includes a compound with the above structural formula 1 and is used for staining microorganisms
  • the second dye includes a compound with the following chemical formula and is used for staining cells:
  • the embodiments of the present invention can simultaneously realize the detection of microorganisms in the blood and the detection of nucleated red blood cells through one detection of the same blood sample to be tested.
  • Example 19 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples to be tested using flow cytometry
  • Example 20 Use of the cyanine dye of the present invention in identifying microorganisms in blood samples to be tested using flow cytometry
  • the first dye includes a compound with the above structural formula 3 and is used for staining microorganisms
  • the second dye includes a compound with the following chemical formula and is used for staining cells:
  • Dot plot the presence of microorganisms in the blood sample to be tested can be identified based on the first scatter plot shown in Figure 35A, and the leukocytes, nucleated cells in the blood sample to be tested can be identified based on the second scatter plot shown in Figure 35B red blood cells and basophils and count them.
  • the embodiments of the present invention can simultaneously realize the detection of microorganisms in the blood and the detection of nucleated red blood cells through one detection of the same blood sample to be tested.
  • Example 21 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples to be tested using flow cytometry
  • a certain amount of Pseudomonas Klebsiella was added to the physiological saline to obtain a simulated body fluid sample to be tested; the above simulated body fluid sample to be tested was tested using the reagents and methods of Example 20 to obtain forward The scattered light intensity FSC and the first fluorescence intensity FL1; the first scatter plot shown in Figure 36 is generated according to the forward scattered light intensity information and the first fluorescence intensity; it can be identified based on the first scatter plot shown in Figure 36 The presence of microorganisms in simulated body fluid samples.
  • Example 22 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry
  • embodiments of the present invention can simultaneously detect parasites in blood and detect nucleated red blood cells through one detection of the same blood sample to be tested.
  • Example 23 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry
  • the first dye includes a compound with the above-mentioned structural formula 2 and is used for staining parasites
  • the second dye includes a compound with the following chemical formula and is used for staining cells:
  • Fluorescence intensity FL2 generate a first scatter plot as shown in Figure 38A based on the forward scattered light intensity information and the first fluorescence intensity, and generate a first scatter plot as shown in Figure 38B based on the forward scattered light intensity information and the second fluorescence intensity.
  • the second scatter plot shown in Figure 38A can identify the presence of malaria parasites in the blood sample to be tested based on the first scatter plot shown in Figure 38A, and the blood sample to be tested can be identified based on the second scatter plot shown in Figure 38B white blood cells, nucleated red blood cells, and basophils and count them.
  • embodiments of the present invention can simultaneously detect parasites in blood and detect nucleated red blood cells through one detection of the same blood sample to be tested.
  • Example 24 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry
  • embodiments of the present invention can simultaneously detect parasites in blood and detect nucleated red blood cells through one detection of the same blood sample to be tested.
  • Example 25 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry
  • the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously detect parasites and white blood cells in blood through one detection of the same blood sample to be tested.
  • Example 26 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry
  • the first dye includes a compound with the structural formula 3 in the above Table 1 and is used for staining parasites
  • the second dye includes a compound with the following chemical formula and is used for staining cells:
  • a sample liquid to be tested is formed for measurement; then, a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm is used to test the sample liquid to be tested to obtain the forward scattered light intensity FSC, side scattered light intensity SSC, The first fluorescence intensity FL1 and the second fluorescence intensity FL2; generate a first scatter plot as shown in Figure 41A according to the forward scattered light intensity information and the first fluorescence intensity, and generate a first scatter plot as shown in Figure 41A according to the side scattered light intensity information and the second The fluorescence intensity generates a second scatter plot as shown in Figure 41B; based on the first scatter plot as shown in Figure 41A it is possible to identify the presence of Plasmodium in the blood sample to be tested, and based on the second scatter plot as shown in Figure 41B
  • the graph can classify the white blood cells in the blood sample to be tested into four categories, and obtain the white blood cell classification results shown in Table 5.
  • the same blood sample to be tested was tested using the DIFF channel (using Mindray BC-6800's DIFF supporting reagent) on the existing blood analyzer (Mindray, model BC-6800), and the leukocyte classification shown in Table 5 was obtained. result.
  • the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously detect parasites and white blood cells in blood through one detection of the same blood sample to be tested.
  • Example 27 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry
  • the first dye includes a compound with the above-mentioned structural formula 4 and is used for staining parasites
  • the second dye includes a compound with the following chemical formula and is used for staining cells:
  • a sample liquid to be tested is formed for measurement; then, a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm is used to test the sample liquid to be tested to obtain the forward scattered light intensity FSC, side scattered light intensity SSC, The first fluorescence intensity FL1 and the second fluorescence intensity FL2; generate a first scatter plot as shown in Figure 42A according to the forward scattered light intensity information and the first fluorescence intensity, and generate a first scatter plot as shown in Figure 42A according to the side scattered light intensity information and the second The fluorescence intensity generates a second scatter plot as shown in Figure 42B; based on the first scatter plot as shown in Figure 42A, the presence of malaria parasites in the blood sample to be tested can be identified, and based on the second scatter plot as shown in Figure 42B
  • the graph can classify the white blood cells in the blood sample to be tested into four categories, and obtain the white blood cell classification results shown in Table 6.
  • the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously detect parasites and white blood cells in blood through one detection of the same blood sample to be tested.

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Abstract

涉及荧光染料，特别涉及花菁染料、其制备方法及其用途。具体地，提供了具有如通式I所示结构的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，其中，R1、R2、R3如说明书所定义。提供的花菁染料具有稳定性好、生物穿透力好等优点。

Description

花菁染料、其制备方法、用途、样本分析方法及分析仪 技术领域
本申请涉及一种荧光染料及其应用，特别涉及花菁染料、其制备方法及其用途、样本分析方法和样本分析仪。本申请还涉及包含所述花菁染料的缀合物以及用于生物样品染色的组合物。
背景技术
DNA(DeoxyriboNucleic Acid，脱氧核糖核酸)是一类带有遗传信息的生物大分子。生物体正常细胞一般具有比较稳定的DNA二倍体含量，而当发生病变时，会发生异常改变；且不同种类生物体的DNA含量一般有所不同。因此对DNA的特异性识别和精确测量，尤其是在活细胞内进行检测，具有重大意义。利用荧光染料进行DNA的定性或定量分析因具有灵敏度高、响应快等优点而引起广大科研工作者的兴趣。
目前，商品化的染料主要有菲啶类、吖啶类、咪唑类和花菁家族类等。然而，这些染料应用过程都存在着一定的局限性。其一，较大一部分染料与DNA结合后呈现荧光淬灭，导致在荧光成像等可视化应用中实用价值不高。其二，当前大部分染料的专一性较差，无法特异性地与DNA相结合，极大地限制其应用潜力。其三，目前多数染料本身有很大的毒性和致癌性，且需通过增大细胞膜的通透性才能对进行生物样品标记。然而，这种通透性处理对细胞和生物组织造成损害，限制了在活细胞中的应用。
在众多荧光染料中，花菁类荧光染料因其波长范围宽，摩尔消光系数大，荧光量子产率适中等优点，作为生物分子荧光探针等已被广泛的应用。尽管花菁类染料中的部分已被商品化，但这些染料大部分分子较大，结构复杂，活细胞通透性低。此外，大多数染料的斯托克位移较小，导致激发光谱与发射光谱之间严重串扰，造成背景干扰和荧光自淬灭现象，限制了其应用。
微生物为自然界中存在的体型细小、结构简单、肉眼看不见的微小生物，其须借助于特殊仪器放大后才能观察到。微生物包括原核类(如细菌、放线菌、支原体、立克次氏体、衣原体、螺旋体)、真核类(如真菌、原生动物、藻类)、非细胞类(病毒和朊病毒)。
很多疾病与微生物相关。病原微生物引起的相关疾病包括细菌性、病毒性、真菌性及原生动物引发的疾病。病原微生物可造成血液感染性疾病，包括菌血症、败血症、毒血症、脓毒血症等。其中，败血症是一种全身性的感染综合征(SIRS)，可造成感染性休克及多器官功能障碍，其死亡率高达30-50％。由此可见，微生物的快速检测意义重大。
目前，临床上诊断微生物的金标准为血培养。然而，对于血培养而言，从血到平板培养到出结果整个过程操作繁琐，且耗时非常长(通常需要3-5天以上)，往往等到结果出来后病人病情已经非常严重。基于此，各种新型有效的检测手段相继涌现。这些检测手段包括免疫检测法和电化学检测法等。
免疫检测法是指利用抗原-抗体的特异性反应进行特定微生物的检测，包括传统的凝集素试验法和沉淀试验法以及新型的酶联免疫吸附法(ELISA)、放射性标记法及化学发光免疫法等。免疫检测法的特异性非常强，检测速度也比传统培养法快。然而，由于存在抗体难以获得、抗体成本高、检测灵敏度不足、反应活性容易受环境影响等缺点，免疫检测法难以在临床微生物检测中广泛应用。
电化学检测法主要是利用微生物代谢过程产生的电信号变化来检测微生物，常见的方法包括阻抗分析法、电位分析法、电流分析法等。基于电化学检测法的设备小、成本低，但是灵敏度不足。
血细胞寄生微小生物是指寄生于动物血液或血细胞中的寄生虫，如疟疾或弓形虫等。血液中的寄生虫的发病周期较短，危害性较高，因此对血细胞中的寄生虫的早期准确诊断是有效的疾病管理和监测所必需的。
目前主要有三种方法能够实现检测血细胞寄生微小生物。第一种方法是通过对血涂片进行显微检查来对血细胞寄生微小生物进行的准确检测和定量，然而这高度依赖于操作人员的训练和技能。第二种方法是基于抗原-抗体反应进行的快速诊断测试(RDT)，该方法不需要太高的操作技能，但是通常较为昂贵的，并且对于低水平血细胞寄生虫缺乏足够的灵敏度。第三种方法是在利用血细胞分析仪进行血常规筛查时，实现检测血细胞中的寄生虫，这种方法简单易操作、成本较低并且不依赖于操作人员的训练和技能。
然而，目前利用血细胞分析仪检测血液中的寄生虫的准确性仍然不高。
发明内容
为了解决上述问题，本申请的发明人通过分子修饰，获得了新的苯并噻唑类的花菁染料，其热稳定性有较明显的改善，而且相比于现有苯并噻唑类的花菁染料，本发明的花菁染料具有高的生物穿透力，更适合用于细胞染料及成像。
化合物
在第一方面，本申请提供了具有如通式I所示结构的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，
其中，
R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基；
R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基；
Y不存在或为抗衡阴离子；
并且限定：
当R3为氢时，R1和R2不同时为甲基且R1和R2不同时为苄基。
本发明的化合物中，R1和R2可以相同，也可以不同。在一些实施方案中，R1和R2独立地选自C1-6直链烷基、C1-6直链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基。
在一些实施方案中，R1和R2中的至少一个为C1-18直链或支链亚烷基-磺酸基。
在一些实施方案中，R1和R2不同且独立地选自C1-6直链烷基、苄基、C1-6直链亚烷基-羧基、C1-6直链亚烷基-磺酸基、C1-6直链亚烷基-巯基、C1-6直链亚烷基-氨基。
在一些实施方案中，R1和R2相同且选自C1-6直链烷基、C1-6直链亚烷基-磺酸基、C1-6直链亚烷基-羧基。
在一些实施方案中，R3选自氢、磺酸基、卤素、氰基、C1-6烷基。
在一些实施方案中，通式I中的Y为抗衡阴离子，此时，Y可以选自卤素离子(例如F-、Cl-、Br-、I-)、ClO4 -、PF6 -、CF3SO3 -、BF4 -、乙酸根、甲磺酸根或对甲苯磺酸根。
在一些实施方案中，通式I中的Y不存在，此时，所述化合物可以为内盐。“内盐”在本领域中还被称为“两性离子”。在一些实施方案中，本发明的化合物可以在分子内同时含有酸性基团(例如磺酸基或羧基)和碱性基团(例如氨基或噻唑环)，所述酸性基团和碱性基团互相中和而生成内盐。
应当理解的是，当化合物分子中含有多个酸性基团和/或多个碱性基团时，各酸性基团和/或各碱性基团都可能作为成盐基团。化合物由各种成盐方式形成的盐均包含在本发明的范围内。
在一些实施方案中，R1和R2中的至少一个选自C1-18直链或支链亚烷基-磺酸基、C1-18直链或支链亚烷基-羧基。
本发明的化合物可以具有以下任一结构：

在一些实施方案中，本发明的化合物为上述结构式5、6或9代表的化合物。
缀合物和用于生物样品染色的组合物
本发明的化合物及其水合物、溶剂化物、立体异构体、互变异构体或晶型可以直接用于生物样品的染色，也可以衍生物的形式使用，所述衍生物包括但不限于缀合物。
本文中使用的“缀合物”是指本发明的化合物、其水合物、溶剂化物、立体异构体、互变异构体或晶型通过共价键与其它分子连接而形成的化合物。可与本发明的化合物、其水合物、溶剂化物、立体异构体、互变异构体或晶型缀合的分子可为与细胞或细胞成分特异性结合的分子，包括但不限于抗体、抗原、受体、配体、酶、底物、辅酶等。通常，测试样品与荧光缀合物温育一段时间，使得该荧光缀合物与测试样品中的某些细胞或细胞成分特异性结合，该荧光缀合物与细胞或细胞成分的结合也可被称为染色。该染色步骤可依次进行多次，或用多种缀合物同时进行多种染色。染色完成后，样品在包含激发光源和测定装置的分析仪器中进行分析，其中激发光源激发缀合物中的本发明荧光染料，而测定装置测定由激发的荧光染料产生的发射光。
本申请还提供了一种用于生物样品染色的组合物，其中所述组合物包含本发明的化合物、其水合物、溶剂化物、立体异构体、互变异构体或晶型、或本发明的缀合物。在一些实施方案中，所述生物样品为核酸。在一些实施方案中，所述生物样品为脱氧核糖核酸。
用途
本申请还提供了本发明的化合物、其水合物、溶剂化物、立体异构体、互变异构体或晶型、或本发明的缀合物、或本发明的组合物在对生物样品进行染色中的用途。在一些实 施方案中，所述生物样品为核酸。在一些实施方案中，所述生物样品为脱氧核糖核酸。
本申请还提供了本发明的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型、或本发明的缀合物、或本发明的组合物在采用流式细胞术识别待测血液样本中的寄生虫中的用途。在一些实施方案中，所述寄生虫选自：蛔虫、钩虫、绦虫、阴道毛滴虫、肝吸虫、卫斯特曼氏并殖吸虫、弓形虫、猪囊虫、旋毛虫、阿米巴虫、杜氏利什曼原虫、疟原虫、血吸虫、丝虫、包虫、疥螨、毛囊螨、虱子、跳蚤。
本申请还提供了本发明的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型、或本发明的缀合物、或本发明的组合物在采用流式细胞术识别待测血液样本或待测体液样本中的微生物中的用途。
在一些实施方案中，所述微生物选自：
细菌(例如金黄色葡萄球菌、大肠杆菌、铜绿假单胞杆菌、痢疾杆菌、百日咳杆菌、白喉杆菌、脑膜炎双球菌、结核分枝杆菌、破伤风梭状杆菌、麻风杆菌、A组溶血性链球菌、布鲁氏菌、霍乱杆菌、伤寒杆菌、炭疽杆菌、淋病奈瑟菌、霍乱弧菌、克雷伯氏假单孢菌、以及副伤寒甲、乙或丙沙门杆菌)，
病毒(例如流感病毒、腮腺炎病毒、风疹病毒、乙脑病毒、登革病毒、流行性出血热病毒、狂犬病毒、人乳头状病毒、脊髓灰质炎病毒、麻疹病毒、水痘带状疱疹病毒、肝炎病毒、新型肠道病毒70型、柯萨奇病毒A24型变种、人免疫缺陷病毒、痘病毒(例如猴痘病毒))，
真菌(例如白色念珠菌、红色毛癣菌、絮状表皮癣菌)，
支原体(例如肺炎支原体、溶脲脲原体、人型支原体、生殖器支原体)，
衣原体(例如沙眼衣原体、肺炎衣原体、鹦鹉衣原体、家畜衣原体)，
立克次氏体(例如普氏立克次氏体、莫氏立克次氏体、立克次氏立克次氏体、恙虫病立克次氏体)，
放线菌(例如以色列放线菌)，
螺旋体(例如钩端螺旋体、梅毒螺旋体)。
在一些实施方案中，血液或体液是指来自哺乳动物、尤其是人类的血液或体液。其中，体液(body fluid)根据部位的不同可分为尿液、汗液、脑脊液(cerebrospinal fluid)、浆膜腔液(serous cavity fluid)、关节滑膜液(synovial fluid)等。正常人体上述体液均有少量存在。浆膜腔中积液包括胸腔积液(pleural effusion)、腹腔积液(ascites)和心包积液(pericardial effusion)，关节腔中的积液为滑膜液(joint effusion)。
通用合成方法
本发明化合物可通过本领域的通用方法合成得到。示例性地，本发明的苯并噻唑类化合物可以通过以下方法合成：首先由未取代或取代的甲基苯并噻唑开始，使其与R2X(X为F、Cl、Br或I)加热回流反应，得到季铵盐形式的中间物I。随后将连接分子4-羟基间苯二甲醛与所述中间物I加热回流反应(所述中间物I与4-羟基间苯二甲醛的摩尔比大于或等于2)，使得中间体I与连接分子缩合，得到本发明的苯并噻唑类化合物。
当R2为C1-18直链或支链亚烷基-磺酸基时，中间物I也可以通过以下方法合成：由未取代或取代的甲基苯并噻唑等原料开始，使其与合适的磺内酯通过开环反应获得中间物I。
以上方法适合于R1和R2相同的情况。
当R1和R2不相同时，本发明的苯并噻唑类化合物可以通过以下示例性的方法合成：将R1或R2取代的甲基苯并噻唑与连接分子4-羟基间苯二甲醛加热回流反应(4-羟基间苯二甲醛与R1或R2取代的甲基苯并噻唑的摩尔比大约为2)，得到含甲酰基的中间物I’；使所得中间物I’与R2或R1取代的甲基苯并噻唑发生缩合反应，得到本发明的苯并噻唑类化合物。
上述方法中，各中间物或产物可通过本领域公知的分离纯化技术回收，以达到需要的纯度。
上述方法中使用的各种原料均可市售获得，或者可通过本领域技术人员公知的方法或现有技术中公开的方法由本领域公知的原料制备得到。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的实验操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“C1-18直链或支链烷基”是指含有1-18个碳原子的直链或支链烷烃去掉一个氢原子后得到的基团，其具体实例包括但不限于：甲基、乙基、正丙基、正丁基、正戊基、正己基、异丙基、叔丁基、异丁基、正庚基、正辛基、正壬基、正癸基、正十一烷基、正十二烷基、正十三烷基、正十四烷基、正十五烷基、正十六烷基、正十七烷基、正十八烷基。
如本文中所使用的，“C1-6烷基”是指含有1-6个碳原子的直链或支链烷烃去掉一个氢原子后得到的基团，其具体实例包括但不限于：甲基、乙基、正丙基、正丁基、正戊基、正己基、异丙基、叔丁基、异丁基等。
如本文中所使用的，“C1-6直链烷基”是指含有1-6个碳原子的直链烷烃去掉一个氢原子后得到的基团，其具体实例包括但不限于：甲基、乙基、正丙基、正丁基、正戊基、正己基。
C1-18直链或支链亚烷基是指含有1-18个碳原子的直链或支链烷烃去掉两个氢原子后得到的基团，其具体实例包括但不限于亚甲基、亚乙基、亚丙基、亚丁基等。术语“C1-6直链亚烷基”是指含有1-6个碳原子的直链烷烃去掉两个氢原子后得到的基团。
如本文中所使用的，术语“卤素”包括氟、氯、溴和碘。
如本文中所使用的，术语“卤代”是指基团或化合物上的氢被一个或多个卤素原子取代，包括全卤代和部分卤代。
如本文中所使用的，术语“C1-6烷氧基”是指，以C1-6烷基-O-方式形成的基团。
如本文中所使用的，术语“溶剂化物”(或“溶剂合物”)是指化合物与有机溶剂(例如甲醇、乙醇、丙醇、乙腈等)分子缔合形成的物质。
如本文中所使用的，术语“水合物”是指化合物与水分子缔合形成的物质。
如本文中所使用的，术语“晶型”是指物质的晶体结构。物质在结晶时由于受各种因素影响，使分子内或分子间键合方式发生改变，致使分子或原子在晶格空间排列不同，形成不同的晶体结构。本发明化合物可以一种晶体结构存在，也可以多种晶体结构存在，即具有“多晶型”。本发明化合物可以不同的晶型存在。
如本文中所使用的，术语“立体异构体”包括构象异构体和构型异构体，其中所述构 型异构体主要包括顺反异构体和旋光异构体。本发明化合物可以以立体异构体的形式存在，并因此涵盖所有可能的立体异构体形式，及其任何组合或任何混合物。例如单一对映异构体，单一非对映异构体或以上的混合物。当本发明化合物含有烯烃双键时，除非特别说明，否则其包括顺式异构体和反式异构体，以及其任何组合。
本发明所述的化合物可以以互变异构体形式存在，其通过一个或多个双键位移而具有不同的氢的连接点。例如，酮和它的烯醇形式是酮-烯醇互变异构体。应当理解，本发明包含所有化合物的酮-烯醇式互变异构体。各互变异构体及其混合物都包括在本发明的范围中。
发明的有益效果
与现有技术相比，本发明的荧光染料具有以下一个或多个有益效果：
1、相比于现有染料，本发明的染料的热稳定性有较明显的改善；
2、染料用于细胞染色需要较高的细胞穿透力，相比于现有染料，本发明的染料具有高的生物穿透力，更适合用于细胞染料及成像；
3、本发明的染料可以特异性地与DNA结合，有利于对DNA的特异性识别和精确测量；
4、本发明的染料具有良好的活细胞通透性，能够在不破坏细胞膜的情况下进入细胞对核酸进行染色，毒性小且致癌性低；
5、本发明的染料的激发光为波长较小的蓝绿色光，能够识别微小颗粒，提高了对小粒子的检测能力；
6、本发明的染料能够使用普通绿色半导体激光器作为光源，大大降低了使用成本；
7、本发明的染料结构简单，制备其的原料易得，合成产率高，易于实现产业化。
为了解决上述问题，本发明的另一个任务在于提供一种样本分析方法和样本分析仪，其能够以流式细胞术为基础在使用荧光标记技术的情况下准确、灵敏、低成本且快速地识别血液或体液中的微生物。
为此，本发明第二方面提出一种样本分析方法，包括下列步骤：
采用包含第一染料的染料试剂和用于溶解红细胞的溶血剂处理同一份待测生物样本，以获得待测样本液，其中，所述待测生物样本为待测血液样本或待测体液样本，所述第一染料能对微生物进行染色；
使所述待测样本液中的粒子逐个通过光学检测区并用光照射流过所述光学检测区的粒子，以获得所述待测样本液中的粒子在被光照射之后所产生的散射光信息和荧光信息，所述荧光信息包括来自所述第一染料的第一荧光信息；以及
基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物。
在一些实施方案中，基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物包括：
基于所述散射光信息和所述第一荧光信息生成第一散点图；并且
基于所述第一散点图识别所述待测样本液中的微生物。
在一些实施方案中，所述散射光信息包括前向散射光信息；并且
基于所述散射光信息和所述第一荧光信息产生第一散点图包括：基于所述前向散射光信息和所述第一荧光信息生成所述第一散点图。
在一些实施方案中，基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物包括：
从所述第一散点图获取微生物特征区域和白细胞区域，所述微生物特征区域的第一荧光信息的强度大于所述白细胞区域的第一荧光信息的强度；并且
基于所述微生物特征区域识别所述待测样本液中的微生物。
在一些实施方案中，基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物包括：
基于所述散射光信息和所述第一荧光信息获取所述待测样本液中的微生物的数量。
在一些实施方案中，所述待测生物样本为待测血液样本，所述样本分析方法还包括：
基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫、尤其是疟原虫。
在一些实施方案中，所述待测生物样本为待测血液样本，所述染料试剂还包括不同于第一染料的第二染料，所述荧光信息还包括来自第二染料的第二荧光信息，其中，所述第二染料能对血液中的白细胞进行染色；并且
所述样本分析方法还包括：基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞分类结果。
在一些实施方案中，所述散射光信息包括侧向散射光信息和前向散射光信息；
基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物包括：基于所述前向散射光信息和所述第一荧光信息识别所述待测样本液中的微生物；并且
基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞分类结果包括：基于所述侧向散射光信息和所述第二荧光信息获取所述待测样本液的白细胞分类结果。
在一些实施方案中，所述待测生物样本为待测血液样本，所述染料试剂还包括不同于第一染料的第二染料，所述荧光信息还包括来自第二染料的第二荧光信息，其中，第二染料能对血液中的白细胞和有核红细胞进行染色；并且
所述样本分析方法还包括：基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
在一些实施方案中，所述散射光信息包括前向散射光信息；
基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物包括：基于所述前向散射光信息和所述第一荧光信息识别所述待测样本液中的微生物；并且
基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个包括：基于所述前向散射光信息和所述第二荧光信息获取所述待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
在一些实施方案中，用光照射流过所述光学检测区的粒子包括：用单一波长的光照射流过所述光学检测区的粒子。
在一些实施方案中，所述第一染料为能与脱氧核糖核酸特异性结合的染料。
在一些实施方案中，所述第一染料包括具有上述通式I的结构的化合物，其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基，R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基，Y不存在或为抗衡阴离子。
关于该化合物的更多细节和优点请参考以上描述，在此不再赘述。
本发明第三方面提出一种样本分析仪，包括：
采样装置，用于定量吸取待测生物样本，所述待测生物样本为待测血液样本或待测体液样本；
样本制备装置，具有反应池和试剂供应部，其中，所述反应池用于接收所述采样装置所吸取的所述待测生物样本，以及接收所述试剂供应部提供的包含第一染料的染料试剂和用于溶解红细胞的溶血剂，所述采样装置所吸取的所述待测生物样本与所述试剂供应部提供的染料试剂和溶血剂在所述反应池中混合，以制备成待测样本液，其中，所述第一染料能对微生物进行染色；
光学检测装置，包括光源、流动室、散射光检测器和荧光检测器，所述光源用于发射光束以照射所述流动室，所述流动室与所述反应池连通并且所述待测样本液中的粒子可逐个通过所述流动室，所述散射光检测器用于检测通过所述流动室的粒子在被光照射后产生的散射光信息，所述荧光检测器用于检测通过所述流动室的粒子在被光照射后产生的荧光信息，所述荧光信息包括来自所述第一染料的第一荧光信息；以及
处理器，被配置为从所述光学检测装置获取所述散射光信息和所述荧光信息，并且基于所述散射光信息和所述第一荧光信息识别待测样本液中的微生物。
在一些实施方案中，所述处理器还被配置为在基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物时执行下列步骤：
基于所述散射光信息和所述第一荧光信息生成第一散点图；并且
基于所述第一散点图识别所述待测样本液中的微生物。
在一些实施方案中，所述散射光信息包括前向散射光信息；并且
所述处理器还被配置为基于所述前向散射光信息和所述第一荧光信息生成所述第一散点图。
在一些实施方案中，所述处理器还被配置为：
从所述第一散点图获取微生物特征区域和白细胞区域，所述微生物特征区域的第一荧光信息的强度大于所述白细胞区域的第一荧光信息的强度；并且
基于所述微生物特征区域识别所述待测样本液中的微生物。
在一些实施方案中，所述处理器还被配置为在基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物时执行下列步骤：
基于所述散射光信息和所述第一荧光信息获取所述待测样本液中的微生物的数量。
在一些实施方案中，所述待测生物样本为待测血液样本，所述处理器还被配置为：
基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫、尤其是疟原虫。
在一些实施方案中，所述待测生物样本为待测血液样本，所述染料试剂还包括不同于第一染料的第二染料，所述第二染料能对血液中的白细胞进行染色，所述荧光信息还包括来自第二染料的第二荧光信息；并且
所述处理器还被配置为：基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞分类结果。
在一些实施方案中，所述散射光信息包括侧向散射光信息和前向散射光信息；并且
所述处理器还被配置为：基于所述前向散射光信息和所述第一荧光信息识别所述待测 样本液中的微生物，以及基于所述侧向散射光信息和所述第二荧光信息获取所述待测样本液的白细胞分类结果。
在一些实施方案中，所述待测生物样本为待测血液样本，所述染料试剂还包括不同于第一染料的第二染料，所述第二染料能对血液中的白细胞和有核红细胞进行染色，所述荧光信息还包括来自第二染料的第二荧光信息；并且
所述处理器还被配置为：基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
在一些实施方案中，所述散射光信息包括前向散射光信息；并且
所述处理器还被配置为：基于所述前向散射光信息和所述第一荧光信息识别所述待测样本液中的微生物，以及基于所述前向散射光信息和所述第二荧光信息获取所述待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
在一些实施方案中，所述光源被构造为用单一波长的光照射所述流动室。
在一些实施方案中，所述第一染料为能与脱氧核糖核酸特异性结合的染料。
在一些实施方案中，所述第一染料包括具有上述通式I的结构的化合物，其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基，R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基，Y不存在或为抗衡阴离子。
关于该化合物的更多细节和优点请参考以上描述，在此不再赘述。
在本发明第二方面提出的样本分析方法和第三方面提出的样本分析仪中，首先采用能对微生物进行染色的染料和用于溶解红细胞的溶血剂处理待测血液样本或待测体液样本，以获得待测样本液，然后采用流式细胞术获取待测样本液的散射光信息和荧光信息，基于所述散射光信息和所述荧光信息能够识别在待测样本液中是否存在微生物。由此能够准确、灵敏、低成本且快速地检测血液或体液中的微生物。
为了解决上述问题，本发明的又另一个任务在于提供一种血液分析方法和血液分析仪，其能够以流式细胞术为基础在使用荧光标记技术的情况下，在低成本血常规检测过程中简单、灵敏、快速且准确地检测血液中的寄生虫。
为此，本发明第四方面提供一种血液分析方法，包括下列步骤：
采用包含第一染料的染料试剂和用于溶解红细胞的溶血剂处理同一份待测血液样本，以获得待测样本液，其中，所述第一染料能对血液中的寄生虫进行染色，并且所述第一染料包括具有上述通式Ⅰ的结构的化合物，其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基，R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基，Y不存在或为抗衡阴离子；
使所述待测样本液中的粒子逐个通过光学检测区并用光照射流过所述光学检测区的粒子，以获得所述待测样本液中的粒子在被光照射之后所产生的散射光信息和荧光信息，所述荧光信息包括来自所述第一染料的第一荧光信息；以及
基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫。
在一些实施方案中，基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫包括：
基于所述散射光信息和所述第一荧光信息生成第一散点图；并且
基于所述第一散点图识别所述待测样本液中的寄生虫。
在一些实施方案中，所述散射光信息包括前向散射光信息；并且
基于所述散射光信息和所述第一荧光信息产生第一散点图包括：基于所述前向散射光信息和所述第一荧光信息生成所述第一散点图。
在一些实施方案中，基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫包括：
从所述第一散点图获取寄生虫特征区域和白细胞区域，所述寄生虫特征区域的第一荧光信息的强度大于所述白细胞区域的第一荧光信息的强度；并且
基于所述寄生虫特征区域识别所述待测样本液中的寄生虫。
在一些实施方案中，基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫包括：
基于所述散射光信息和所述第一荧光信息获取所述待测样本液中的寄生虫的数量。
在一些实施方案中，所述血液分析方法还包括：
基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物。
在一些实施方案中，可以使用根据本发明第一方面所述的化合物作为第一染料所包含的化合物，关于该化合物的更多细节和优点请参考以上描述，在此不再赘述。
在一些实施方案中，所述染料试剂还包括不同于第一染料的第二染料，所述第二染料能对血液中的白细胞和有核红细胞进行染色，所述荧光信息还包括来自第二染料的第二荧光信息；并且
所述血液分析方法还包括：基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
在一些实施方案中，所述散射光信息包括前向散射光信息；
基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫包括：基于所述前向散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫；并且
基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个包括：基于所述前向散射光信息和所述第二荧光信息获取所述待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
在一些实施方案中，所述染料试剂还包括不同于第一染料的第二染料，所述第二染料能对血液中的白细胞进行染色，所述荧光信息还包括来自第二染料的第二荧光信息；并且
所述血液分析方法还包括：基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞分类结果。
在一些实施方案中，所述散射光信息包括侧向散射光信息和前向散射光信息；
基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫包括：基于所述前向散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫；并且
基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞分类结果包括：基于所述侧向散射光信息和所述第二荧光信息获取所述待测样本液的白细胞分类结果。
在一些实施方案中，用光照射流过所述光学检测区的粒子包括：用单一波长的光照射流过所述光学检测区的粒子。
本发明第五方面提供一种血液分析仪，包括：
采样装置，用于定量吸取待测血液样本；
样本制备装置，具有反应池和试剂供应部，其中，所述反应池用于接收所述采样装置所吸取的所述待测血液样本，以及接收所述试剂供应部提供的包含第一染料的染料试剂和用于溶解红细胞的溶血剂，所述采样装置所吸取的所述待测血液样本与所述试剂供应部提供的染料试剂和溶血剂在所述反应池中混合，以制备成待测样本液，其中，所述第一染料包括具有上述通式Ⅰ的结构的化合物，其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基，R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基，Y不存在或为抗衡阴离子；
光学检测装置，包括光源、流动室、散射光检测器和荧光检测器，所述光源用于发射光束以照射所述流动室，所述流动室与所述反应池连通并且所述待测样本液中的粒子可逐个通过所述流动室，所述散射光检测器用于检测通过所述流动室的粒子在被光照射后产生的散射光信息，所述荧光检测器用于检测通过所述流动室的粒子在被光照射后产生的荧光信息，所述荧光信息包括来自所述第一染料的第一荧光信息；以及
处理器，被配置为从所述光学检测装置获取所述散射光信息和所述荧光信息，并且基于所述散射光信息和所述第一荧光信息识别待测样本液中的寄生虫。
在一些实施方案中，所述处理器还被配置为在基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫时执行下列步骤：
基于所述散射光信息和所述第一荧光信息生成第一散点图；并且
基于所述第一散点图识别所述待测样本液中的寄生虫。
在一些实施方案中，所述散射光信息包括前向散射光信息；并且
所述处理器还被配置为基于所述前向散射光信息和所述第一荧光信息生成所述第一散点图。
在一些实施方案中，所述处理器还被配置为：
从所述第一散点图获取寄生虫特征区域和白细胞区域，所述寄生虫特征区域的第一荧光信息的强度大于所述白细胞区域的第一荧光信息的强度；并且
基于所述寄生虫特征区域识别所述待测样本液中的寄生虫。
在一些实施方案中，所述处理器还被配置为在基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫时执行下列步骤：
基于所述散射光信息和所述第一荧光信息获取所述待测样本液中的寄生虫的数量。
在一些实施方案中，所述处理器还被配置为：
基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物。
在一些实施方案中，可以使用根据本发明第一方面所述的化合物作为第一染料所包含的化合物，关于该化合物的更多细节和优点请参考以上描述，在此不再赘述。
在一些实施方案中，所述染料试剂还包括不同于第一染料的第二染料，所述第二染料能对血液中的白细胞和有核红细胞进行染色，所述荧光信息还包括来自第二染料的第二荧光信息；并且
所述处理器还被配置为：基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
在一些实施方案中，所述散射光信息包括前向散射光信息；并且
所述处理器还被配置为：基于所述前向散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫，以及基于所述前向散射光信息和所述第二荧光信息获取所述待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
在一些实施方案中，所述染料试剂还包括不同于第一染料的第二染料，所述第二染料能对血液中的白细胞进行染色，所述荧光信息还包括来自第二染料的第二荧光信息；并且
所述处理器还被配置为：基于所述散射光信息和所述第二荧光信息获取所述待测样本液的白细胞分类结果。
在一些实施方案中，所述散射光信息包括侧向散射光信息和前向散射光信息；并且
所述处理器还被配置为：基于所述前向散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫，以及基于所述侧向散射光信息和所述第二荧光信息获取所述待测样本液的白细胞分类结果。
在一些实施方案中，所述光源被构造为用单一波长的光照射所述流动室。
在本发明第四方面提出的样本分析方法和第五方面提出的样本分析仪中，首先采用能对寄生虫进行染色的花菁染料、尤其是苯并噻唑类的花菁染料和用于溶解红细胞的溶血剂处理待测血液样本，以获得待测样本液，然后采用流式细胞术获取待测样本液的散射光信息和荧光信息，基于所述散射光信息和所述荧光信息能够识别在待测样本液中是否存在寄生虫。由此能够准确、灵敏、低成本且快速地检测血液中的寄生虫。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1为按照本发明的用于识别微生物的样本分析方法的一种实施例的示意流程图。
图2为按照本发明一实施例的在待测生物样本为血液样本时的第一散点图。
图3为按照本发明一实施例的在待测生物样本为体液样本时的第一散点图。
图4为按照本发明的用于识别微生物的样本分析方法的另一种实施例的示意流程图。
图5为按照本发明的在对同一份血液样本的一次检测中同时获得微生物识别结果和白细胞分类结果的散点图。
图6为按照本发明的用于识别微生物的样本分析方法的又另一种实施例的示意流程图。
图7为按照本发明的在对同一份血液样本的一次检测中同时获得微生物识别结果和有核红细胞识别结果的散点图。
图8为按照本发明一实施例的两种染料的发射光谱的示意图。
图9为按照本发明一实施例的一种大斯托克斯位移染料的发射光谱与激发光谱的示意图。
图10为按照本发明的用于识别寄生虫的血液分析方法的一种实施例的示意流程图；
图11为按照本发明一实施例的用于识别寄生虫的第一散点图；
图12为按照本发明的用于识别寄生虫的血液分析方法的另一种实施例的示意流程图；
图13为按照本发明的在对同一份血液样本的一次检测中同时获得寄生虫识别结果和有核红细胞识别结果的散点图；
图14为按照本发明的用于识别寄生虫的血液分析方法的又另一种实施例的示意流程图；
图15为按照本发明的在对同一份血液样本的一次检测中同时获得寄生虫识别结果和白细胞分类结果的散点图；
图16为按照本发明的样本分析仪的一种实施例的结构示意图。
图17至19为按照本发明的光学检测装置的不同实施例的结构示意图。
图20显示了实施例7中染料随DNA浓度增加，荧光光谱的变化情况。
图21显示了实施例7中染料随RNA浓度增加，荧光光谱的变化情况。
图22为实施例7中荧光强度与小牛胸腺DNA和RNA浓度的线性关系图。
图23显示了实施例8中激光显微镜下观察化合物II对HepG2活细胞的染色的结果。
图24显示了实施例9中激光显微镜下观察化合物III对HepG2活细胞的染色的结果。
图25显示了实施例10中染料稳定性的评价结果。
图26显示了实施例11中染料细胞穿透力的评价结果。
图27为按照本发明实施例12在对同一份血液样本的一次检测中同时获得微生物识别结果和白细胞分类结果的散点图。
图28为按照本发明实施例13对体液样本进行测试得到的散点图。
图29为按照本发明实施例14在对同一份血液样本的一次检测中同时获得微生物识别结果和白细胞分类结果的散点图。
图30为按照本发明实施例15对体液样本进行测试得到的散点图。
图31为按照本发明实施例16在对同一份血液样本的一次检测中同时获得微生物识别结果和白细胞分类结果的散点图。
图32为按照本发明实施例17对体液样本进行测试得到的散点图。
图33为按照本发明实施例18在对同一份血液样本的一次检测中同时获得微生物识别结果和有核红细胞识别结果的散点图。
图34为按照本发明实施例19对体液样本进行测试得到的散点图。
图35为按照本发明实施例20在对同一份血液样本的一次检测中同时获得微生物识别结果和有核红细胞识别结果的散点图。
图36为按照本发明实施例21对体液样本进行测试得到的散点图。
图37为按照本发明实施例22在对同一份血液样本的一次检测中同时获得寄生虫识别结果和有核红细胞识别结果的散点图。
图38为按照本发明实施例23在对同一份血液样本的一次检测中同时获得寄生虫识别结果和有核红细胞识别结果的散点图。
图39为按照本发明实施例24在对同一份血液样本的一次检测中同时获得寄生虫识别结果和有核红细胞识别结果的散点图。
图40为按照本发明实施例25在对同一份血液样本的一次检测中同时获得寄生虫识别结果和白细胞分类结果的散点图。
图41为按照本发明实施例26在对同一份血液样本的一次检测中同时获得寄生虫识别结果和白细胞分类结果的散点图。
图42为按照本发明实施例27在对同一份血液样本的一次检测中同时获得寄生虫识别结果和白细胞分类结果的散点图。
具体实施方式
下面将结合附图对本发明实施例进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，本发明实施例所涉及的术语“第一\第二\第三”仅仅是区别类似的对象，不代表针对对象的特定排序，可以理解地，“第一\第二\第三”在允许的情况下可以互换特定的顺序或先后次序。
作为现代临床血液科血常规分析的必备仪器，以流式细胞术为基本原理的血细胞分析仪(也称血液分析仪、血球仪)对血细胞的检测具有快速、准确、操作极其简单等优点，是现代临床检验中不可或缺的全自动化血液细胞快速检测的仪器。
基于此，本发明提出了一种基于流式细胞术和荧光标记技术的用于快速检测血液或体液中的病原微生物的技术方案。
在本发明实施例中，微生物是指肉眼难以看清且需要借助光学显微镜或电子显微镜才能观察到的微小生物。微生物包括细菌、病毒、真菌和少数藻类等。
如图1所示，本发明实施例首先提供一种基于流式细胞术和荧光标记技术快速检测血液或体液中的微生物的样本分析方法100，样本分析方法100包括下列步骤S110、S120和S130。
在步骤S110中，采用包含第一染料的染料试剂和用于溶解红细胞的溶血剂处理同一份待测生物样本，以获得待测样本液。其中，待测生物样本为待测血液样本或待测体液样本。在此，第一染料能对微生物进行染色。也就是说，当待测血液样本或待测体液样本中存在微生物时，使用第一染料处理待测血液样本或待测体液样本能够使得其中的微生物被染色。
例如，在步骤S110中，可以将溶血剂和染料试剂先后加入到同一份待测血液样本或待测体液样本中，以得到待测样本液，然后使待测样本液孵育，使得染料试剂能够充分地染色待测样本液中的待测物质。再例如，在步骤S110中，也可以先将染料试剂预先与溶血剂混合得到混合试剂，然后将混合试剂与待测生物样本以250：1-1000：1体积比混合，混合均匀后，使混合得到的待测样本液在25℃-50℃的温度下孵育10秒-1分钟、优选孵育20秒-40秒。
可以理解的，在本发明实施例中，溶血剂用于溶解血液或体液中的红细胞，将红细胞裂解为碎片，但能够保持白细胞的形态基本不变。
在一些实施例中，溶血剂可以包含阳离子表面活性剂、非离子表面活性剂、阴离子表面活性剂、两亲性表面活性剂、缓冲对中的任意一种或几种的组合。阳离子表面活性剂例如选自十二烷基三甲基氯化铵、辛基三甲基溴化铵、十四烷基三甲基氯化铵中的至少一种或几种的组合。非离子表面活性剂例如选自长链脂肪醇聚氧乙烯、烷基酚聚氧乙烯醚、脂肪酸聚氧乙烯醚、脂肪胺聚氧乙烯醚中的至少一种或几种的组合。缓冲对例如选自磷酸盐类、柠檬酸盐类、Tris-HCl中的至少一种或几种的组合。阴离子表面活性剂例如选自十二烷基苯磺酸、脂肪醇酰硫酸钠、乙氧基化脂肪酸甲酯磺酸钠、仲烷基磺酸钠、醇醚羧酸盐 中的至少一种或几种的组合。
在另一些实施例中，溶血剂可以包括烷基糖苷、三萜皂苷、甾族皂苷中的至少一种。
在步骤S120中，使待测样本液中的粒子逐个通过光学检测区并用光照射流过光学检测区的粒子，以获得待测样本液中的粒子在被光照射之后所产生的散射光信息和荧光信息。在此，荧光信息至少包括来自第一染料的第一荧光信息，也就是说，第一荧光信息包括待测样本液中的粒子与第一染料结合后在光激发下所产生的荧光信号。
也就是说，在步骤S120中，基于流式细胞术原理获得待测样本液的散射光信息和荧光信息。当光、例如激光束照射流过光学检测区的粒子时，粒子本身的特性(如体积、染色程度、细胞内容物大小及含量、细胞核密度等)可阻挡或改变激光束的方向，从而产生与其特征相应的各种角度的散射光，这些散射光经信号检测器接收后可以获得与粒子结构和组成相关的光信息、即本发明的散射光信息和荧光信息。在此，散射光信息例如包括前向散射光信息和侧向散射光信息中的至少一种。其中，前向散射光反应粒子的数量和体积，侧向散射光(Side scatter，SS)反应细胞内部结构(如细胞内颗粒或细胞核)的复杂程度，以及荧光(Fluorescence，FL)反应细胞中核酸物质的含量。利用这些光信息能够识别待测样本液中的各类粒子。
在本发明实施例中，散射光信息包括散射光信号强度，并且荧光信息包括荧光信号强度。
在步骤S130中，基于散射光信息和第一荧光信息识别待测样本液中的微生物。
在一些实施例中，步骤S130、即基于散射光信息和第一荧光信息识别待测样本液中的微生物可以包括：基于散射光信息和第一荧光信息生成第一散点图；并且基于第一散点图识别待测样本液中的微生物。
在本发明实施例中，散点图可以是二维散点图或三维散点图，其上分布有多个粒子的二维或三维特征信息，其中，散点图的X坐标轴、Y坐标轴和Z坐标轴均表征每个粒子的一种特性。例如，在一个散点图中，X坐标轴表征前向散射光信号强度，Y坐标轴表征荧光信号强度，Z轴坐标轴表征侧向散射光信号强度。应说明的是，本文中的散点图不受图形形式的限制，也可以是数据形式，比如与散点图具有等同或相近分辨率的表格或列表的数字形式呈现，或者采用任何本领域已知的其他适合的方式呈现。
进一步地，散射光信息可以包括前向散射光信息FSC、尤其是前向散射光信号强度。相应地，基于散射光信息和第一荧光信息产生第一散点图可以包括：基于前向散射光信息FSC、尤其是前向散射光信号强度和第一荧光信息FL1、尤其是第一荧光信号强度生成第一散点图，如图2和图3所示。其中，图2示出在待测生物样本为血液样本时的第一散点图，并且图3示出在待测生物样本为体液样本时的第一散点图。
进一地，如图2和图3所示，基于散射光信息和第一荧光信息识别待测样本液中的微生物可以包括：
从第一散点图获取微生物特征区域P1和白细胞区域P2，微生物特征区域的第一荧光信息的强度大于白细胞区域的第一荧光信息的强度；并且
基于微生物特征区域P1识别待测样本液中的微生物。
在此，通过发明人的反复研究，在以前向散射光信息为横坐标并且以第一荧光信息为纵坐标的散点图、例如图2和图3所示的二维散点图中，在白细胞群(即白细胞区域)的上方存在某一特定区域(即该特定区域的第一荧光信号强度不小于白细胞区域的第一荧光 信号强度)，当血液样本或体液样本中存在微生物时，在该特征区域中总是存在成团的散点，而当血液样本或体液样本中不存在微生物时，在该特征区域中不存在成团的散点。因此，在该特征区域中成团的散点可以用于表征微生物粒子团，该特征区域也可以称为微生物特征区域。当该特征区域中的散点的数量超过预定阈值时，则认为在待测血液样本或体液样本中存在微生物。
在一些实施例中，基于散射光信息和第一荧光信息识别待测样本液中的微生物可以包括：基于散射光信息、尤其是前向散射光信息和第一荧光信息获取待测样本液中的微生物的数量。例如，在图2和图3所示的实施例中，微生物特征区域P1中的散点的数量可以用于表征待测样本液中的微生物的数量。
在一些可行的具体示例中，可以预先收集大量含有微生物的生物样本的散点数据(例如微生物特征区域P1中的散点的数量)和这些生物样本中的微生物的实际数量，通过拟合得到微生物的散点数据与实际数量的相关性曲线，从而得到相应的计算模型、例如线性计算模型。在待测生物样本的实际测试中，根据本发明的方法获取待测生物样本的微生物散点数据，基于待测生物样本的微生物散点数据和上述预先确定的计算模型，可估算待测生物样本中的微生物的数量，从而实现微生物的定量分析。
在一些实施例中，当所述待测生物样本为待测血液样本时，所述样本分析方法100还可以包括：基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫、尤其是疟原虫，尤其是基于所述第一散点图识别所述待测样本液中的寄生虫。由此能够通过对同一份待测生物样本的一次检测同时实现血液中的微生物和寄生虫检测，在不增加用血量的情况下提高血液检测效率并且节省试剂成本。
在一些实施例中，当所述待测生物样本为待测血液样本时，在步骤S110中，所述染料试剂可以进一步包括不同于第一染料的第二染料，该第二染料能对血液中的细胞进行染色。此时，在步骤S120中获得的荧光信息还包括来自第二染料的第二荧光信息FL2、尤其是第二荧光信号强度，即，第二荧光信息包括待测样本液中的粒子与第二染料结合后在光激发下所产生的荧光信号。由此能够进一步根据散射光信息和第二荧光信息获取待测样本液的细胞参数、例如白细胞参数、有核红细胞参数等。
作为一些实现方式，在步骤S110中使用能对血液中的白细胞进行染色的第二染料、尤其是用于白细胞分类的第二染料。相应地，如图4所示，样本分析方法100还可以包括步骤S140：基于散射光信息和第二荧光信息获取待测样本液的白细胞分类结果。在一个具体的示例中，可以基于散射光信息和第二荧光信息将待测样本液中的白细胞分类为淋巴细胞群、单核细胞群、中性粒细胞群、嗜酸性粒细胞群，并对各类白细胞进行计数，以得到淋巴细胞计数和/或淋巴细胞计数占白细胞计数的比例、单核细胞计数和/或单核细胞计数占白细胞计数的比例、中性粒细胞计数和/或中性粒细胞计数占白细胞计数的比例、嗜酸性粒细胞计数和/或嗜酸性粒细胞计数占白细胞计数的比例。在另一个示例中，也可以基于散射光信息和第二荧光信息将待测样本液中的白细胞分类为淋巴细胞群、单核细胞群、中性粒细胞群、嗜酸性粒细胞群和嗜碱性粒细胞群，并对各类白细胞进行计数。由此能够通过对同一份待测血液样本的一次检测同时实现血液中的微生物检测和白细胞检测，在不增加用血量的情况下提高血液检测效率并且节省试剂成本。
在一些实施例中，用于白细胞分类的第二染料可以是能与细胞内的核酸物质结合的核酸染料、例如包括花菁类阳离子化合物，其更多细节可参考申请人于2019年9月28日提 交的中国专利申请CN101750274A，其全部公开内容通过引用合并于此。
在一些实施例中，用于白细胞分类的第二染料是非DNA或RNA特异性的核酸染料，例如包括具有如下化学式Ⅱ的化合物。
进一步地，散射光信息可以包括侧向散射光信息SSC和前向散射光信息FSC。相应地，基于散射光信息和第一荧光信息识别所述待测样本液中的微生物、即步骤S130可以包括：基于前向散射光信息FSC和第一荧光信息FL1识别待测样本液中的微生物，如图5A所示；并且基于散射光信息和第二荧光信息获取待测样本液的白细胞分类结果、即步骤S140可以包括：基于侧向散射光信息SSC和第二荧光信息FL2获取待测样本液的白细胞分类结果、例如如上所述的白细胞四分类结果，如图5B所示。例如，根据侧向散射光信息和第二荧光信息生成第二散点图，在该第二散点图上根据设门技术将待测样本液中的白细胞分为中性粒细胞群、淋巴细胞群、单核细胞群和嗜酸性粒细胞群并对这些细胞群进行计数。
进一步地，步骤S140也可以包括基于前向散射光信息FSC、侧向散射光信息SSC和第二荧光信息FL2获取待测样本液的白细胞分类结果。
作为另一些实现方式，在步骤S110中使用能对血液中的有核细胞、例如白细胞和有核红细胞进行染色的第二染料，该第二染料尤其是能用于识别有核红细胞。相应地，如图6所示，样本分析方法100还可以包括步骤S150：基于散射光信息和第二荧光信息获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个，尤其是基于散射光信息和第二荧光信息获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数。由此能够通过对同一份待测血液样本的一次检测同时实现血液中的微生物检测以及白细胞和/或有核红细胞检测，在不增加用血量的情况下提高血液检测效率并且节省试剂成本。
在一些实施例中，用于对有核细胞染色的第二染料也可以是能与细胞内的核酸物质结合的核酸染料，但是不同于上述用于白细胞分类的染料，也可以是能与细胞内的蛋白物质结合的蛋白染料。在一个示例中，用于对有核细胞染色的第二染料例如包括具有如下化学式Ⅲ的化合物。
进一步地，散射光信息可以包括前向散射光信息FSC。相应地，基于散射光信息和第一荧光信息识别待测样本液中的微生物、即步骤S130可以包括：基于前向散射光信息FSC 和第一荧光信息FL1识别所述待测样本液中的微生物，如图7A所示；并且基于散射光信息和第二荧光信息获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个、即步骤S150可以包括：基于前向散射光信息FSC和第二荧光信息FL2获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个，如图7B所示。
在一些实施例中，在使用包括第一染料和第二染料的染料试剂处理待测生物样本的情况下，在步骤S120中，用光照射流过所述光学检测区的粒子包括：用单一波长的光、尤其是蓝光、例如具有450纳米左右的波长的蓝光照射流过光学检测区的粒子。
在此，在采用一个激发光同时激发两种染料、即第一染料和第二染料的情况下，为了更好地区分收集与第一染料对应的第一荧光信息和与第二染料对应的第二荧光信息，从而更加准确地在溶血条件下通过两种染料来区分血液中的白细胞和微生物，这样选择第一染料和第二染料，使得第一染料与第二染料的发射光谱的峰值所对应的波长之差的绝对值大于30纳米且小于80纳米。备选地或附加地，这样选择第一染料和第二染料，使得第一染料与第二染料的发射光谱的重叠量不大于50％。通过选择这样的第一染料和第二染料，能够大大减少第一荧光信号和第二荧光信号的相互间的检测干扰、即大大增加第一荧光信号和第二荧光信号区分度。
图8示出了第一染料和第二染料的发射光谱的示意图，虚线表示的曲线为第一染料的发射光谱210，实线表示的曲线为第二染料的发射光谱220。其中，第一染料的发射光谱210的峰值点为A，第二染料的发射光谱220的峰值点为D。在此，峰值点A与峰值点D各自的横坐标的绝对差值(即峰值对应的波长之差)大于30纳米且小于80纳米。此外，第一染料的发射光谱210与第二染料的发射光谱220的重叠量可以为第一多边形面积与第二多边形面积之比，其中，第一多边形面积等于点E、点G以及点C这三点围成的曲边多边形面积，而第二多边形面积等于第一染料的发射光谱210(或第二染料的发射光谱220)与基准线230所围成的曲边多边形面积，其中，基准线230是如图8所示的与横轴平行的虚线横线，该虚线横线处于第一染料的发射光谱210与第二染料的发射光谱220归一化峰值的5％处。点E和点F分别是第二染料的发射光谱220与基准线230的左交点和右交点，点B和点C分别是第一染料的发射光谱210与基准线230的左交点和右交点。在此，第一染料的发射光谱210与第二染料的发射光谱220的重叠量不大于50％。
进一步有利的是，尤其是在采用单一波长的光照射的情况下，第一染料与第二染料的发射光谱的峰值所对应的波长之差的绝对值大于40且小于80纳米、优选大于50纳米且小于80纳米、更优选大于50纳米且小于70纳米，从而能够进一步减少第一荧光信号和第二荧光信号的相互间的检测干扰。
此外有利的是，第一染料与第二染料的发射光谱的重叠量不大于35％、优选不大于15％，由此也能够进一步减少第一荧光信号和第二荧光信号的相互间的检测干扰。其中，第一染料与第二染料的发射光谱的重叠量越小，越有利于区分第一荧光信号和第二荧光信号。
接下来描述本发明的第一染料的一些实施方式。
在一些实施例中，第一染料可以是大斯托克斯位移染料。在此，大斯托克斯位移染料是指发射光谱与激发光谱各自的峰值所对应的波长之差大于预定阈值的染料。
图9为一种大斯托克斯位移染料的光谱示意图，该大斯托克斯位移染料的激发光谱(也称为吸收光谱)310通过虚线示出，发射光谱320通过实线示出。其中，激发光谱310 的峰值点为A1，发射光谱320的峰值点为A2。峰值点A2与峰值点A1各自的横坐标的差值(即发射光谱与激发光谱各自的峰值对应的波长之差)大于预定阈值。该预定阈值例如可以大于30纳米且小于150纳米、优选大于50纳米且小于100纳米。
通过使用大斯托克斯位移染料尤其是能够减少第一荧光信号和第二荧光信号的相互间的检测干扰。
作为一些实现方式，第一染料为能与脱氧核糖核酸(即DNA)特异性结合的染料、例如花菁染料、尤其是苯并噻唑类的花菁染料。
在一些实施例中，第一染料可以包括具有通式I的结构的化合物：
其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基；R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基；并且Y不存在或为抗衡阴离子。
在此，本发明提出的包括具有通式I的结构的化合物的第一染料具有以下一个或多个优点：热稳定性较好；具有高的生物(微生物、细胞)穿透力；能够特异性地与DNA结合，有利于对DNA的特异性识别和精确测量；具有良好的活细胞通透性，能够在不破坏细胞膜的情况下进入细胞对核酸进行染色，毒性小且致癌性低；能够使用波长较小的蓝绿色光来激发，从而能够识别微小颗粒，提高了对小粒子的检测能力；能够使用普通绿色半导体激光器作为光源，大大降低了使用成本；结构简单，制备其的原料易得，合成产率高，易于实现产业化。
在一些实施例中，R1和R2独立地选自C1-6直链烷基、C1-6直链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基。
在一些实施例中，R1和R2中的至少一个为C1-18直链或支链亚烷基-磺酸基、C1-18直链或支链亚烷基-羧基。
在一些实施例中，R1和R2不同且独立地选自C1-6直链烷基、苄基、C1-6直链亚烷基-羧基、C1-6直链亚烷基-磺酸基、C1-6直链亚烷基-巯基、C1-6直链亚烷基-氨基。在另一些实施例中，R1和R2相同且选自C1-6直链烷基、C1-6直链亚烷基-磺酸基、C1-6直链亚烷基-羧基。
在一些实施例中，R3选自氢、磺酸基、卤素、氰基、C1-6烷基。
在一些实施例中，当R3为氢时，R1和R2不同时为甲基且R1和R2不同时为苄基。
在一些实施例中，通式I中的Y为抗衡阴离子，此时，Y可以选自卤素离子(例如F-、Cl-、Br-、I-)、ClO4 -、PF6 -、CF3SO3 -、BF4 -、乙酸根、甲磺酸根或对甲苯磺酸根。在另一些实施例中，通式I中的Y不存在，此时，所述化合物可以为内盐。“内盐”在本领域中还被称为“两性离子”。例如，本发明的化合物可以在分子内同时含有酸性基团(例如磺酸基或 羧基)和碱性基团(例如氨基或噻唑环)，所述酸性基团和碱性基团互相中和而生成内盐。
应当理解的是，当化合物分子中含有多个酸性基团和/或多个碱性基团时，各酸性基团和/或各碱性基团都可能作为成盐基团。化合物由各种成盐方式形成的盐均包含在本发明的范围内。
在一些实施例中，本发明的具有通式I的结构的化合物可以具有下表所示的任一结构：

优选的，本发明的化合物为上述结构式5、6或9代表的化合物。
在另一些实施例中，在通式I中，R1和R2各自独立地选自由C1-18烷基、C1-18磺酸基，C1-18羧基、C1-18羟基、C1-18NR5R6、苄基和取代苄基组成的组，其中，所述取代苄基的取代基选自由C1-18烷基、CN、COOH、NH2、NO2、OH、SH、C1-6烷氧基、C1-6烷基氨基、C1-6酰氨基、卤素和C1-6卤代烷基组成的组，优选R1和R2为相同C1-18磺酸基；R3选自由H、C1-18磺酸基、苯基、OR6和卤素组成的组；并且Y-为负离子。
在一些实施例中，本发明的染料试剂可以保存于甘油、甘醇、乙二醇等水溶性有机相中。
在一些实施例中，本发明的染料试剂可以单独保存，也可以与溶血剂混合保存。
本发明还提出了一种基于流式细胞术和荧光标记技术的用于快速检测血液中的寄生虫的技术方案。
在本发明实施例中，寄生虫选自：蛔虫、钩虫、绦虫、阴道毛滴虫、肝吸虫、卫斯特曼氏并殖吸虫、弓形虫、猪囊虫、旋毛虫、阿米巴虫、杜氏利什曼原虫、疟原虫、血吸虫、丝虫、包虫、疥螨、毛囊螨、虱子、跳蚤。
如图10所示，本发明实施例还提供一种基于流式细胞术和荧光标记技术快速检测血液中的寄生虫的血液分析方法1000，血液分析方法1000包括下列步骤S1100、S1200和S1300。
在步骤S1100中，采用包含第一染料的染料试剂和用于溶解红细胞的溶血剂处理同一份待测血液样本，以获得待测样本液。在此，第一染料能对血液中的寄生虫进行染色。也就是说，当待测血液样本中存在寄生虫时，使用第一染料处理待测血液样本能够使得其中的寄生虫被染色。在此，第一染料为花菁染料、尤其是苯并噻唑类的花菁染料并且包括具有通式Ⅰ的结构的化合物：
其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基，R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基，Y不存在或为抗衡阴离子。
例如，在步骤S1100中，可以将溶血剂和染料试剂先后加入到同一份待测血液样本中，以得到待测样本液，然后使待测样本液孵育，使得染料试剂能够充分地染色待测样本液中的待测物质。在在例如，在步骤S1100中，也可以先将染料试剂预先与溶血剂混合得到混 合试剂，然后将混合试剂与待测血液样本以250：1-1000：1体积比混合，混合均匀后，使混合得到的待测样本液在25℃-50℃的温度下孵育10秒-1分钟、优选孵育20秒-40秒。
以上在方法100的步骤S110中所使用的溶血剂的各个实施例可相应地应用于在步骤S1100中所使用的溶血剂，在此不再赘述。
以上在方法100的步骤S110中所使用的具有通式Ⅰ的结构的化合物的各个实施例可相应地应用于在步骤S1100中所使用的第一染料的化合物，在此不再赘述。
在步骤S1200中，使待测样本液中的粒子逐个通过光学检测区并用光照射流过光学检测区的粒子，以获得待测样本液中的粒子在被光照射之后所产生的散射光信息和荧光信息。在此，荧光信息至少包括来自第一染料的第一荧光信息，也就是说，第一荧光信息包括待测样本液中的粒子与第一染料结合后在光激发下所产生的荧光信号。
也就是说，在步骤S1200中，基于流式细胞术原理获得待测样本液的散射光信息和荧光信息。当光、例如激光束照射流过光学检测区的粒子时，粒子本身的特性(如体积、染色程度、细胞内容物大小及含量、细胞核密度等)可阻挡或改变激光束的方向，从而产生与其特征相应的各种角度的散射光，这些散射光经信号检测器接收后可以获得与粒子结构和组成相关的光信息、即本发明的散射光信息和荧光信息。在此，散射光信息例如包括前向散射光信息和侧向散射光信息中的至少一种。其中，前向散射光反应粒子的数量和体积，侧向散射光(Side scatter，SS)反应细胞内部结构(如细胞内颗粒或细胞核)的复杂程度，以及荧光(Fluorescence，FL)反应细胞中核酸物质的含量。利用这些光信息能够识别待测样本液中的各类粒子。
在本发明实施例中，散射光信息包括散射光信号强度，并且荧光信息包括荧光信号强度。
在步骤S1300中，基于散射光信息和第一荧光信息识别待测样本液中的寄生虫。
在一些实施例中，步骤S1300、即基于散射光信息和第一荧光信息识别待测样本液中的寄生虫可以包括：基于散射光信息和第一荧光信息生成第一散点图；并且基于第一散点图识别待测样本液中的寄生虫。
进一步地，散射光信息可以包括前向散射光信息FSC、尤其是前向散射光信号强度。相应地，基于散射光信息和第一荧光信息产生第一散点图可以包括：基于前向散射光信息FSC、尤其是前向散射光信号强度和第一荧光信息FL1、尤其是第一荧光信号强度生成第一散点图，如图11所示。
进一地，如图11所示，基于散射光信息和第一荧光信息识别待测样本液中的寄生虫可以包括：
从第一散点图获取寄生虫特征区域P1和白细胞区域P2，寄生虫特征区域的第一荧光信息的强度大于白细胞区域的第一荧光信息的强度；并且
基于寄生虫特征区域P1识别待测样本液中的寄生虫。
在此，通过发明人的反复研究，在以前向散射光信息为横坐标并且以第一荧光信息为纵坐标的散点图、例如图11所示的二维散点图中，在白细胞群(即白细胞区域)的上方存在某一特定区域(即该特定区域的第一荧光信号强度不小于白细胞区域的第一荧光信号强度)，当血液样本中存在寄生虫时，在该特征区域中总是存在成团的散点，而当血液样本中不存在寄生虫时，在该特征区域中不存在成团的散点。因此，在该特征区域中成团的散点可以用于表征寄生虫粒子团，该特征区域也可以称为寄生虫特征区域。当该特征区域中的 散点的数量超过预定阈值时，则认为在待测血液样本中存在寄生虫。
在一些实施例中，基于散射光信息和第一荧光信息识别待测样本液中的寄生虫可以包括：基于散射光信息、尤其是前向散射光信息和第一荧光信息获取待测样本液中的寄生虫的数量。例如，在图11所示的实施例中，寄生虫特征区域P1中的散点的数量可以用于表征待测样本液中的寄生虫的数量。
在一些可行的具体示例中，可以预先收集大量含有寄生虫的血液样本的散点数据(例如寄生虫特征区域P1中的散点的数量)和这些血液样本中的寄生虫的实际数量，通过拟合得到寄生虫的散点数据与实际数量的相关性曲线，从而得到相应的计算模型、例如线性计算模型。在待测血液样本的实际测试中，根据本发明的方法获取待测血液样本的寄生虫散点数据，基于待测血液样本的寄生虫散点数据和上述预先确定的计算模型，可估算待测血液样本中的寄生虫的数量，从而实现寄生虫的定量分析。
在一些实施例中，所述血液分析方法1000还可以包括：基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物，尤其是基于所述第一散点图识别所述待测样本液中的微生物。由此能够通过对同一份待测血液样本的一次检测同时实现血液中的微生物和寄生虫检测，在不增加用血量的情况下提高血液检测效率并且节省试剂成本。
在一些实施例中，本发明的染料试剂可以保存于甘油、甘醇、乙二醇等水溶性有机相中。
在一些实施例中，本发明的染料试剂可以单独保存，也可以与溶血剂混合保存。
在一些实施例中，在步骤S1100中，所述染料试剂可以进一步包括不同于第一染料的第二染料，该第二染料能对血液中的细胞进行染色。此时，在步骤S1200中获得的荧光信息还包括来自第二染料的第二荧光信息FL2、尤其是第二荧光信号强度，即，第二荧光信息包括待测样本液中的粒子与第二染料结合后在光激发下所产生的荧光信号。由此能够进一步根据散射光信息和第二荧光信息获取待测样本液的细胞参数、例如白细胞参数、有核红细胞参数等。
作为一些实现方式，在步骤S1100中使用能对血液中的有核细胞、例如白细胞和有核红细胞进行染色的第二染料，该第二染料尤其是能用于识别有核红细胞。相应地，如图12所示，血液分析方法1000还可以包括步骤S1400：基于散射光信息和第二荧光信息获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个，尤其是基于散射光信息和第二荧光信息获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数。由此能够通过对同一份待测血液样本的一次检测同时实现血液中的寄生虫检测以及白细胞和/或有核红细胞检测，在不增加用血量的情况下提高血液检测效率并且节省试剂成本。
在一些实施例中，用于对有核细胞染色的第二染料可以是能与细胞内的核酸物质结合的核酸染料，也可以是能与细胞内的蛋白物质结合的蛋白染料。在一个示例中，用于对有核细胞染色的第二染料例如包括具有如下化学式Ⅲ的化合物。
进一步地，散射光信息可以包括前向散射光信息FSC。相应地，基于散射光信息和第一荧光信息识别待测样本液中的寄生虫、即步骤S1300可以包括：基于前向散射光信息FSC和第一荧光信息FL1识别所述待测样本液中的寄生虫，如图13A所示；并且基于散射光信息和第二荧光信息获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个、即步骤S1400可以包括：基于前向散射光信息FSC和第二荧光信息FL2获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个，如图13B所示。
作为另一些实现方式，在步骤S1100中使用能对血液中的白细胞进行染色的第二染料、尤其是用于白细胞分类的第二染料。相应地，如图14所示，血液分析方法1000还可以包括步骤S1500：基于散射光信息和第二荧光信息获取待测样本液的白细胞分类结果。在一个具体的示例中，可以基于散射光信息和第二荧光信息将待测样本液中的白细胞分类为淋巴细胞群、单核细胞群、中性粒细胞群、嗜酸性粒细胞群，并对各类白细胞进行计数，以得到淋巴细胞计数和/或淋巴细胞计数占白细胞计数的比例、单核细胞计数和/或单核细胞计数占白细胞计数的比例、中性粒细胞计数和/或中性粒细胞计数占白细胞计数的比例、嗜酸性粒细胞计数和/或嗜酸性粒细胞计数占白细胞计数的比例。在另一个示例中，也可以基于散射光信息和第二荧光信息将待测样本液中的白细胞分类为淋巴细胞群、单核细胞群、中性粒细胞群、嗜酸性粒细胞群和嗜碱性粒细胞群，并对各类白细胞进行计数。由此能够通过对同一份待测血液样本的一次检测同时实现血液中的寄生虫检测和白细胞检测，在不增加用血量的情况下提高血液检测效率并且节省试剂成本。
在一些实施例中，用于白细胞分类的第二染料也可以是能与细胞内的核酸物质结合的核酸染料、例如包括花菁类阳离子化合物，其更多细节可参考申请人于2019年9月28日提交的中国专利申请CN101750274A，其全部公开内容通过引用合并于此。
在一些实施例中，用于白细胞分类的第二染料是非DNA或RNA特异性的核酸染料，例如包括具有如下化学式Ⅱ的化合物。
进一步地，散射光信息可以包括侧向散射光信息SSC和前向散射光信息FSC。相应地，基于散射光信息和第一荧光信息识别所述待测样本液中的寄生虫、即步骤S1300可以包括： 基于前向散射光信息FSC和第一荧光信息FL1识别待测样本液中的寄生虫，如图15A所示；并且基于散射光信息和第二荧光信息获取待测样本液的白细胞分类结果、即步骤S1500可以包括：基于侧向散射光信息SSC和第二荧光信息FL2获取待测样本液的白细胞分类结果、例如如上所述的白细胞四分类结果，如图15B所示。例如，根据侧向散射光信息和第二荧光信息生成第二散点图，在该第二散点图上根据设门技术将待测样本液中的白细胞分为中性粒细胞群、淋巴细胞群、单核细胞群和嗜酸性粒细胞群并对这些细胞群进行计数。
进一步地，步骤S1500也可以包括基于前向散射光信息FSC、侧向散射光信息SSC和第二荧光信息FL2获取待测样本液的白细胞分类结果。
在一些实施例中，在使用包括第一染料和第二染料的染料试剂处理待测血液样本的情况下，在步骤S1200中，用光照射流过所述光学检测区的粒子包括：用单一波长的光、尤其是蓝光、例如具有4500纳米左右的波长的蓝光照射流过光学检测区的粒子。
在此，在采用一个激发光同时激发两种染料、即第一染料和第二染料的情况下，为了更好地区分收集与第一染料对应的第一荧光信息和与第二染料对应的第二荧光信息，从而更加准确地在溶血条件下通过两种染料来区分血液中的白细胞和寄生虫，这样选择第一染料和第二染料，使得第一染料与第二染料的发射光谱的峰值所对应的波长之差的绝对值大于30纳米且小于80纳米。备选地或附加地，这样选择第一染料和第二染料，使得第一染料与第二染料的发射光谱的重叠量不大于50％，参见图8。通过选择这样的第一染料和第二染料，能够大大减少第一荧光信号和第二荧光信号的相互间的检测干扰、即大大增加第一荧光信号和第二荧光信号区分度。
进一步有利的是，尤其是在采用单一波长的光照射的情况下，第一染料与第二染料的发射光谱的峰值所对应的波长之差的绝对值大于40且小于80纳米、优选大于50纳米且小于80纳米、更优选大于50纳米且小于70纳米，从而能够进一步减少第一荧光信号和第二荧光信号的相互间的检测干扰。
此外有利的是，第一染料与第二染料的发射光谱的重叠量不大于35％、优选不大于15％，由此也能够进一步减少第一荧光信号和第二荧光信号的相互间的检测干扰。其中，第一染料与第二染料的发射光谱的重叠量越小，越有利于区分第一荧光信号和第二荧光信号。
在一些实施例中，第一染料可以是大斯托克斯位移染料。在此，大斯托克斯位移染料是指发射光谱与激发光谱各自的峰值所对应的波长之差大于预定阈值的染料。再次参见图9，峰值点A2与峰值点A1各自的横坐标的差值大于预定阈值。该预定阈值例如可以大于30纳米且小于150纳米、优选大于50纳米且小于100纳米。通过使用大斯托克斯位移染料尤其是能够减少第一荧光信号和第二荧光信号的相互间的检测干扰。
本发明提供的血液分析方法1000的其他实施例可参考以上对样本分析方法100的描述。
本发明还提供一种基于流式细胞术和荧光标记技术的样本分析仪400。如图16所示，样本分析仪400包括采样装置410、样本制备装置420、光学检测装置430和处理器440。样本分析仪400还包括液路系统(未示出)，用于连通采样装置410、样本制备装置420及光学检测装置430，以便在这些装置之间进行液体传输。
采样装置410用于定量吸取待测生物样本，其中，待测生物样本为待测血液样本或待测体液样本。例如，采样装置410具有带吸移管嘴的吸移管并且具有驱动装置，该驱动装置用于驱动吸移管通过吸移管嘴定量吸取待测生物样本。采样装置可将所采集的待测生物 样本输送至样本制备装置420。
样本制备装置420具有反应池和试剂供应部。反应池用于接收采样装置410所吸取的待测生物样本，以及接收试剂供应部提供的包含第一染料的染料试剂和用于溶解红细胞的溶血剂，采样装置410所吸取的待测生物样本与试剂供应部提供的染料试剂和溶血剂在反应池中混合，以制备成待测样本液。
根据本发明第一实施方式，第一染料能对微生物进行染色。在该第一实施方式中，第一染料的更多实施例可参考以上在样本分析方法100中所使用的第一染料的各个实施例，在此不再赘述。
根据本发明第二实施方式，第一染料能对寄生虫进行染色并且包括具有通式Ⅰ的结构的化合物：
其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基，R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基，Y不存在或为抗衡阴离子。
在该第二实施方式中，第一染料的更多实施例可参考以上在样本分析方法100中所使用的第一染料的各个实施例以及在血液分析方法1000中所使用的第一染料的各个实施例，在此不再赘述。
光学检测装置430包括光源、流动室、散射光检测器和荧光检测器，光源用于发射光束以照射流动室，流动室与反应池连通并且待测样本液中的粒子可逐个通过流动室，散射光检测器用于检测通过流动室的粒子在被光照射后产生的散射光信息，荧光检测器用于检测通过流动室的粒子在被光照射后产生的荧光信息，荧光信息包括来自第一染料的第一荧光信息。
在本文中，流动室指适于检测光散射信号和荧光信号的聚焦液流的腔室。当一粒子、如一血细胞通过流动室的检测孔时，该粒子将来自光源的被导向该检测孔的入射光束向各方向散射。可以在相对于该入射光束的一个或多个不同角度设置光检测器，以检测被该粒子散射的光，从而得到光散射信号。由于不同的粒子具有不同的光散射特性，因此光散射信号可以用于区分不同的粒子群体。具体地，在入射光束附近所检测的光散射信号通常被称为前向光散射信号或小角度光散射信号。在一些实施例中，该前向光散射信号可以从与入射光束约1°至约10°的角度上进行检测。在其他一些实施例中，该前向光散射信号可以从与入射光束约2°至约6°的角度上进行检测。在与入射光束呈约90°的方向所检测的光散射信号通常被称为侧向光散射信号。在一些实施例中，该侧向光散射信号可以是从与入射光束呈约65°至约115°的角度上进行检测。通常地，来自被荧光染料染色的血细胞所发出的荧光信号一般也在与入射光束呈约90°的方向上进行检测。
在一些实施例中，光学检测装置430包括用于检测前向散射光的前向散射光检测器或者用于检测侧向散射光的侧向散射光检测器。光学检测装置430优选包括前向散射光检测 器和侧向散射光检测器。
图17示出光学检测装置430的一个具体示例。该光学检测装置430具有依次布置在一条直线上的光源401、光束整形组件402、流动室403和前向散射光检测器404。在流动室403的一侧，与所述直线成45°角布置有二向色镜406。通过流动室403中的粒子发出的侧向光，一部分透过二向色镜406，被与二向色镜406成45°角布置在二向色镜106后面的荧光检测器405捕获；另一部分侧向光被二向色镜406反射，被与二向色镜406成45°角布置在二向色镜406前面的侧向散射光检测器407捕获。
处理器440用于对光学检测装置430所收集的光学信号进行处理，以得到所要求的结果，例如可以根据收集的各种光学信号生成二维散点图或三维散点图，并在散点图上根据设门(gating)的方法进行粒子分析。处理器440还可以对中间运算结果或最终运算结果进行可视化处理，然后通过显示装置450显示出来。
在一些实施例，处理器440包括但不限于中央处理器(Central Processing Unit，CPU)、微控制单元(Micro Controller Unit，MCU)、现场可编程门阵列(Field-Programmable Gate Array，FPGA)、数字信号处理器(DSP)等用于解释计算机指令以及处理计算机软件中的数据的装置。例如，处理器440用于执行计算机可读存储介质中的各计算机应用程序，从而使样本分析仪400执行相应的检测流程并实时地分析光学检测装置430所检测的光学信号。
此外，样本分析仪400还包括第一机壳460和第二机壳470。显示装置450例如可以为用户界面。光学检测装置130及处理器140设置在第二机壳170的内部。样本制备装置420例如设置在第一机壳460的内部，显示装置450例如设置在第一机壳460的外表面并且用于显示血液细胞分析仪的检测结果。在其他实施例中，具有显示器的计算机可以与样本分析仪400远程通信连接，该计算机例如安装在远离血液细胞分析仪所在的实验室的地方，例如在医生诊疗室中。
根据本发明第一实施方式，处理器440被配置为从光学检测装置430获取散射光信息和荧光信息，并且基于散射光信息和第一荧光信息识别待测样本液中的微生物。
在第一实施方式的一些变型方案中，处理器440还可以被配置为在基于散射光信息和第一荧光信息识别待测样本液中的微生物时执行下列步骤：
基于散射光信息和第一荧光信息生成第一散点图；并且
基于第一散点图识别所述待测样本液中的微生物。
在第一实施方式的一些变型方案中，散射光信息包括前向散射光信息，此时处理器440还可以被配置为基于前向散射光信息和第一荧光信息生成第一散点图。
在第一实施方式的一些变型方案中，处理器440还可以被配置为：
从第一散点图获取微生物特征区域和白细胞区域，微生物特征区域的第一荧光信息的强度大于白细胞区域的第一荧光信息的强度；并且
基于微生物特征区域识别待测样本液中的微生物。
在第一实施方式的一些变型方案中，处理器440还可以被配置为在基于散射光信息和第一荧光信息识别待测样本液中的微生物时执行下列步骤：基于散射光信息和第一荧光信息获取待测样本液中的微生物的数量。
在第一实施方式的一些变型方案中，处理器440还可以被配置为：基于散射光信息和第一荧光信息识别待测样本液中的寄生虫、尤其是疟原虫。
在第一实施方式的一些变型方案中，所述待测生物样本为待测血液样本，染料试剂可以进一步包括不同于第一染料的第二染料，该第二染料能对血液中的细胞进行染色。此时，光学检测装置430获得的荧光信息还包括来自第二染料的第二荧光信息。由此能够进一步根据散射光信息和第二荧光信息获取待测样本液的细胞参数、例如白细胞参数、有核红细胞参数等。
图18示出光学检测装置430的另一个具体示例。该光学检测装置430具有激光器431、前光组件432、流动室433、前向散射光检测器434、第一二向色镜435、侧向散射光检测器436、第二二向色镜437、第一荧光检测器438和第二荧光检测器439。第一荧光检测器438用于检测通过流动室433的粒子在被光照射后产生的与第一染料对应的第一荧光信号，第二荧光检测器439用于检测通过流动室433的粒子在被光照射后产生的与第二染料对应的第二荧光信号。在此，激光器431、前光组件432、流动室433和前向散射光检测器434沿光轴方向依次布置在光轴上，前光组件配置用于使由激光器431发出的激发光在粒子流动方向上汇聚于流动室433的检测区，使得流过流动室433的检测区的粒子能够产生散射光。在流动室433的一侧，第一二向色镜435与光轴成45°角布置。粒子在流过流动室433的检测区时所产生的侧向光的一部分被第一二向色镜435反射并且被侧向散射光检测器436捕获，而另一部分侧向光透过第一二向色镜435到达第二二向色镜437，第二二向色镜437同样与光轴成45°角地布置在第一二向色镜435下游。透过第一二向色镜435的侧向光的一部分被第二二向色镜437反射并且被第一荧光检测器438捕获，而另一部分透过第二二向色镜437被第二荧光检测器439捕获。
在其他实施例中，如图19所示，与图18所示的光学检测装置不同的是，前向散射光检测器434也可以布置成倾斜于光轴。在光轴上，沿着光轴方向在流动室下游布置有反射镜4341，该反射镜将粒子的前向散射光反射到倾斜于光轴布置的前向散射光检测器434中。
在第一实施方式的一些变型方案中，第二染料为能对血液中的白细胞进行染色的染料，尤其是用于白细胞分类的染料。相应地，处理器440还可以被配置为：基于散射光信息和第二荧光信息获取待测样本液的白细胞分类结果。
进一步地，散射光信息包括侧向散射光信息和前向散射光信息。相应地，处理器440还可以被配置为：基于前向散射光信息和第一荧光信息识别待测样本液中的微生物，以及基于侧向散射光信息和第二荧光信息获取待测样本液的白细胞分类结果。
在第一实施方式的另一些变型方案中，第二染料为能对血液中的白细胞和有核红细胞进行染色的染料，尤其是用于识别有核红细胞的染料。相应地，处理器440还可以被配置为：基于散射光信息和第二荧光信息获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
进一步地，散射光信息包括前向散射光信息。相应地，处理器440还可以被配置为：基于前向散射光信息和第一荧光信息识别待测样本液中的微生物，以及基于前向散射光信息和第二荧光信息获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
优选的是，光源可以被构造为用单一波长的光照射流动室。例如，光学检测装置430仅具有一个发出蓝绿光的激光源、尤其是发出蓝光的激光源。
根据本发明第一实施方式的样本分析仪400的更多实施例和优点可参考以上对本发明的样本分析方法100的描述，在此不再赘述。
根据本发明第二实施方式，处理器440被配置为从光学检测装置430获取散射光信息和荧光信息，并且基于散射光信息和第一荧光信息识别待测样本液中的寄生虫。
在第二实施方式的一些变型方案中，处理器440还可以被配置为在基于散射光信息和第一荧光信息识别待测样本液中的寄生虫时执行下列步骤：
基于散射光信息和第一荧光信息生成第一散点图；并且
基于第一散点图识别所述待测样本液中的寄生虫。
在第二实施方式的一些变型方案中，散射光信息包括前向散射光信息，此时处理器440还可以被配置为基于前向散射光信息和第一荧光信息生成第一散点图。
在第二实施方式的一些变型方案中，处理器440还可以被配置为：
从第一散点图获取寄生虫特征区域和白细胞区域，寄生虫特征区域的第一荧光信息的强度大于白细胞区域的第一荧光信息的强度；并且
基于寄生虫特征区域识别待测样本液中的寄生虫。
在第二实施方式的一些变型方案中，处理器440还可以被配置为在基于散射光信息和第一荧光信息识别待测样本液中的寄生虫时执行下列步骤：基于散射光信息和第一荧光信息获取待测样本液中的寄生虫的数量。
在第二实施方式的一些变型方案中，处理器440还可以被配置为：基于散射光信息和第一荧光信息识别待测样本液中的微生物。
在第二实施方式的一些变型方案中，染料试剂可以进一步包括不同于第一染料的第二染料，该第二染料能对血液中的细胞进行染色。此时，光学检测装置430获得的荧光信息还包括来自第二染料的第二荧光信息。由此能够进一步根据散射光信息和第二荧光信息获取待测样本液的细胞参数、例如白细胞参数、有核红细胞参数等。
在第二实施方式的一些变型方案中，第二染料为能对血液中的白细胞和有核红细胞进行染色的染料，尤其是用于识别有核红细胞的染料。相应地，处理器440还可以被配置为：基于散射光信息和第二荧光信息获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
进一步地，散射光信息包括前向散射光信息。相应地，处理器440还可以被配置为：基于前向散射光信息和第一荧光信息识别待测样本液中的寄生虫，以及基于前向散射光信息和第二荧光信息获取待测样本液的白细胞计数、嗜碱性粒细胞计数和有核红细胞计数中的至少一个。
在第二实施方式的另一些变型方案中，第二染料为能对血液中的白细胞进行染色的染料，尤其是用于白细胞分类的染料。相应地，处理器440还可以被配置为：基于散射光信息和第二荧光信息获取待测样本液的白细胞分类结果。
进一步地，散射光信息包括侧向散射光信息和前向散射光信息。相应地，处理器440还可以被配置为：基于前向散射光信息和第一荧光信息识别待测样本液中的寄生虫，以及基于侧向散射光信息和第二荧光信息获取待测样本液的白细胞分类结果。
优选的是，光源可以被构造为用单一波长的光照射流动室。例如，光学检测装置430仅具有一个发出蓝绿光的激光源、尤其是发出蓝光的激光源。
根据本发明第二实施方式的血液分析仪400的更多实施例和优点可参考以上对本发明的样本分析方法100和血液分析方法1000的描述，在此不再赘述。
本发明还提出DNA特异性染料(即能与脱氧核糖核酸特异性结合的染料)在采用流式 细胞术识别待测血液样本中的微生物中的应用。
在一些实施例中，DNA特异性染料包括具有通式I的结构的化合物：
其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基；R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基；并且Y不存在或为抗衡阴离子。
关于本发明提出的DNA特异性染料的更多实施例和优点可参考以上对本发明的样本分析方法100的描述，在此不再赘述。
本发明还提出DNA特异性染料(即能与脱氧核糖核酸特异性结合的染料)在采用流式细胞术识别待测血液样本中的寄生虫中的应用。其中，该DNA特异性染料包括具有通式Ⅰ的结构的化合物：
其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基；R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基；并且Y不存在或为抗衡阴离子。
关于本发明提出的DNA特异性染料的更多实施例和优点可参考以上对本发明的样本分析方法100和血液分析方法1000的描述，在此不再赘述。
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1化合物I的合成
化合物I的结构可用结构式1表示。
第一步，根据下述反应式I制备化合物(E)-4-2-(5-甲酰基-2-羟基苯乙烯基)苯并噻唑-3-丁基-1-磺酸内盐(结构见反应式I右侧)。
在反应瓶中加入5mL甲醇，并依次加入1.33mmol 4-羟基间苯二甲醛(上海毕得医药科技股份有限公司)，0.67mmol(E)-4-(2-(5-甲酰基-2-羟基苯乙烯基)苯并噻唑-3-基)-丁基-1-磺酸盐和2.66mmol吡啶(上海毕得医药科技股份有限公司)。反应在80℃下搅拌43小时。
反应混合物进行抽滤，滤饼用10mL乙醇洗涤3次，滤饼收集并减压干燥后得到0.44mmol的黄色固体粉末，该黄色固体粉末即为(E)-4-(2-(5-甲酰基-2-羟基苯乙烯基)苯并噻唑-3-基)-丁基-1-磺酸内盐。该反应式I的产率约为33％。
对化合物进行核磁共振测试，结果如下。
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)。
第二步根据下述反应式II制备4-(2-((E)-2-((E)-3-((Z))-2-(3-甲基苯并噻唑-2(3H)-烯基)乙烯基)-6-氧代-1,4-环己二烯-1-基)乙烯基)苯并噻唑-3-基)丁烷磺酸内盐(化合物I)。
向反应瓶中加入5mL乙酸，将0.44mmol第二步得到的(E)-4-(2-(5-甲酰基-2-羟基苯乙烯基)苯并噻唑-3-基)-丁基-1-磺酸内盐(结构见反应式Ⅱ左侧)，0.72mmol 2,3二甲基苯并噻唑盐(上海皓鸿生物医药科技有限公司)和1.58mmol乙酸钠依次加入到反 应瓶中，在110℃下反应12小时。
将反应混合物过滤，滤饼用5mL乙腈/水＝1：1的混合溶液洗涤3次，得粗产品。
将上述粗产品经过制备HPLC纯化后得到约0.01mmol红色固体粉末，该红色固体粉末即为4-(2-((E)-2-((E)-3-((Z))-2-(3-甲基苯并噻唑-2(3H)-烯基)乙烯基)-6-氧代-1,4-环己二烯-1-基)乙烯基)苯并噻唑-3-基)丁烷磺酸内盐(化合物I)。反应式II的产率约为20％。
对产物进行核磁共振测试，结果如下：
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)。
经验证，测试结果符合结构式1的结构。
实施例2化合物II的合成
化合物II的结构可用结构式2表示。
第一步，根据下述反应式I制备2-甲基-3-(丁基磺酸)苯并噻唑盐(结构见反应式I右侧)。
在容器中量取50mL甲苯，将34mmol的苯并噻唑(反应式I左侧)和40mmol的1,4-丁磺内酯(上海皓鸿生物医药科技有限公司)加入到容器中。在氮气保护的条件下回流并搅拌24小时，之后停止反应。
对反应后的混合物进行抽滤，之后使用50mL乙酸乙酯洗涤滤饼3次，得到黄色固体产物即为2-甲基-3-(丁基磺酸)苯并噻唑盐。该反应式I的产率约为62％。
第二步，根据下述反应式II制备化合物II(4-(2-((E)-6-氧代-3-((Z)-2-(3-(4-磺丁基)苯并[d]噻唑-2-亚基)亚乙基)环已-1,4-二烯-1-基)乙烯基)苯并[d]噻唑-3-基)丁磺酸。
量取30mL乙酸放入容器中，将1.7mmol的第一步反应得到的2-甲基-3-(丁基磺酸)苯并噻唑盐(结构见反应式II箭头上方)、0.7mmol的4-羟基间苯二甲醛以及2.2mmol的乙酸钠加入到容器中。在氮气保护及温度为80℃的条件下搅拌反应24小时。
将反应后的混合物倒入事先准备好的50mL石油醚:乙酸乙酯＝5:1的溶液中，之后倒出上清液，得粗产物。将上述粗产物经过10mL乙腈比水等于1:1打浆后得到约0.34mmol的棕色固体粉末，该棕色固体粉末即为化合物II，反应式II的产率约为52％。
对产物进行核磁共振测试，结果如下。
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).
经验证，测试结果符合结构式2的结构。
实施例3化合物VI的合成
化合物VI的结构可用结构式6表示，其中，Y-为碘离子。
第一步，根据下述反应式I制备2,3-二甲基-5-氯苯并噻唑碘化季铵
向反应瓶中加入20mL碘甲烷，接着加入10.9mmol 5-氯-2-甲基苯并噻唑(上海毕得医药科技股份有限公司)，该反应加热到80℃并在此温度下搅拌16小时。
将反应混合物过滤，滤饼用10mL乙酸乙酯洗涤三次。收集滤饼并减压干燥后得到7.35mmol白色粉末固体。该白色粉末固体即为2,3-二甲基-5-氯苯并噻唑碘化季铵(反应式I右侧)。该反应式I的产率约为67％。
对产物进行核磁共振测试，结果如下。
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)
第二步，根据下述反应式II制备5-氯-2-((E)-2-((E)-3-((Z)-2-(5-氯-3-甲基苯并噻唑-2(3H)-亚基)乙烯基)-6-氧代环己-1,4-二烯-1-基)乙烯基-3-甲基苯并噻唑盐(化合物VI)
量取6mL乙酸放入容器中，将1.67mmol的第一步反应得到的5-氯-2,3-二甲基苯并噻唑季铵盐(结构见反应式II左侧)加到0.67mmol的4-羟基间苯二甲醛(上海皓鸿生物医药科技有限公司)和2.2mmol的乙酸钠的混合物中。在110℃的条件下搅拌反应12小时。
待反应完毕，将反应后的混合物过滤，收集滤饼得粗产物。
将上述粗产物加入到20mL乙腈中，在90℃条件下搅拌2小时。过滤收集固体，得到约0.46mmol的棕黑色固体.该棕黑色固体粉末即为5-氯-2-((E)-2-((E)-3-((Z)-2-(5-氯-3-甲基苯并噻唑-2(3H)-亚基)乙烯基)-6-氧代环己-1,4-二烯-1-基)乙烯基-3-甲基苯并噻唑盐(化合物VI)，反应式II的产率约为70％。
对产物进行核磁共振测试，结果如下。
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).
经验证，测试结果符合结构式6的结构。
实施例4化合物VII的合成
化合物VII的结构可用结构式7表示。
第一步，根据下述反应式I制备3-(5-羧酸戊基)-2-甲基苯并噻唑溴化盐(结构见反应式I右侧)。
将67mmol的苯并噻唑(上海皓鸿生物医药科技有限公司)(结构见反应式I左侧)加入到容器中。之后加入73.72mmol的6-溴己酸，待反应体系升温至140℃后，搅拌12小时。之后停止反应，待冷却到室温。
对反应后的混合物过滤，用30mL石油醚洗滤饼三次。得到约45mmol黄色固体，该固体即为3-(5-羧酸戊基)-2-甲基苯并噻唑溴化盐，该反应式I的产率约为78％。
对产物进行核磁共振测试，结果如下。
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).
第二步，根据下述反应式II制备2-((反)-2-((反)-6-氧-3-((Z)-2-(5-羧酸戊基)苯并噻唑-2(3H)-基)乙烯基)环己-1,4-二烯基)乙烯基-3-(5-羧酸戊基)苯并噻唑盐(化合物VII)。
将1.7mmol的第一步反应得到的3-(5-羧酸戊基)-2-甲基苯并噻唑溴化盐和0.7mmol4-羟基间苯二甲醛加入到10mL乙酸中，再加入以及2.2mmol乙酸钠。在温度为110℃的条件下搅拌反应12小时。反应结束后，冷却到室温，静置16小时。之后大量固体析出，过滤得到粗产物。
将上述粗产物经过10mL乙腈搅拌1小时后，再次过滤，滤饼用5mL乙腈洗三次，收集固体并在减压下干燥得到约0.43mmol的棕色固体粉末，该棕色固体粉末即为2-((反)-2-((反)-6-氧-3-((Z)-2-(5-羧酸戊基)苯并噻唑-2(3H)-基)乙烯基)环己-1,4-二烯基)乙烯基-3-(5-羧酸戊基)苯并噻唑盐(化合物VII)，反应式II的产率约为64％。
对产物进行核磁共振测试，结果如下。
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).
经验证，测试结果符合结构式7的结构。
实施例5化合物VIII的合成
化合物VIII的结构可用结构式8表示，其中，Y-为碘离子。
第一步，根据下述反应式I制备2,3-二甲基-5-氟苯并噻唑碘化季铵(结构见反应式I右侧)。
向反应瓶中加入10mL碘甲烷，接着加入9.0mmol 5-氟-2-甲基苯并噻唑(上海皓鸿生物医药科技有限公司)，该反应加热到80℃并在此温度下搅拌12小时。
将反应混合物过滤，滤饼用10mL乙酸乙酯洗涤三次。收集滤饼并减压干燥后得到7.1mmol白色粉末固体B。该白色粉末固体即为2,3-二甲基-5-氟苯并噻唑碘化季铵(结构见反应式I右侧)。该反应式I的产率约为79％。
对产物进行核磁共振测试，结果如下。
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).
经验证，测试结果符合反应式I的产物结构。
第二步，根据下述反应式II制5-氟-2-((E)-2-((E)-3-((Z)-2-(5-氟-3-甲基苯并噻唑-2(3H)-亚基)乙烯基)-6-氧代环己-1,4-二烯-1-基)乙烯基-3-甲基苯并噻唑盐(化合物VIII)
将2mmol的第一步反应得到的2,3-二甲基-5-氟苯并噻唑碘化季铵(结构见反应式II左侧)和0.67mmol的4-羟基间苯二甲醛量加到10mL乙酸中，然后加入2.2mmol的 乙酸钠。在120℃的条件下搅拌反应12小时。
待反应完毕，将反应后的混合物过滤，滤饼用10mL乙腈洗涤3次，收集固体，得到约0.46mmol的棕黑色固体.该棕黑色固体粉末即为5-氟-2-((E)-2-((E)-3-((Z)-2-(5-氟-3-甲基苯并噻唑-2(3H)-亚基)乙烯基)-6-氧代环己-1,4-二烯-1-基)乙烯基-3-甲基苯并噻唑盐(化合物VIII)，反应式II的产率约为95％。
对产物进行核磁共振测试，结果如下。
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).
经验证，测试结果符合结构式8的结构。
实施例6化合物IX的合成
化合物IX的结构可用结构式9表示，其中，Y-为碘离子。
第一步，根据下述反应式I制备2-甲基-5-氰基苯并噻唑(结构见反应式I右侧)。
向反应瓶中加入55mL去离子水，氮气氛围下加入0.29mmol乙酸钾并溶解，接着加入55mL二氧六环，5.7mmol 5-溴-2-甲基苯并噻唑(上海毕得医药科技股份有限公司)，2.85mmol三水合铁氰酸钾和0.57mmol XPhos-Pd-G3。所有原料和试剂加完后，置换成氮气氛围。该反应混合物在100℃下反应1小时。
反应结束后，混合物用60mL水稀释，用200mL乙酸乙酯萃取。有机相用无水硫酸钠干燥30min后过滤，滤液减压浓缩得到黄色油状的产物粗品。粗品利用硅胶柱层析(石油醚/乙酸乙酯＝10:1到5:1)纯化后，得到5.72mmol灰色粉末固体，该固体即为2-甲基-5-氰基苯并噻唑(结构见反应式I右侧)。
对产物进行核磁共振测试，结果如下。
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).
经验证，测试结果符合反应式I的产物结构。
第二步，根据下述反应式II制备2,3-二甲基-5-氰基苯并噻唑碘化季铵(结构见反应式II右侧)。
向反应瓶中加入20mL碘甲烷，接着加入5.7mmol 5-氰基-2-甲基苯并噻唑(成都福瑞斯特科技发展有限公司)，该反应加热到80℃并在此温度下搅拌16小时。
将反应混合物过滤，滤饼用10mL乙酸乙酯洗涤三次。收集滤饼并减压干燥后得到2.18mmol黄色粉末固体，该黄色粉末固体即为2,3-二甲基-5-氰基苯并噻唑碘化季铵(结构见反应式II右侧)。该反应式II的产率约为38％。
对产物进行核磁共振测试，结果如下。
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).
经验证，测试结果符合反应式II的产物结构。
第三步，根据下述反应式III制备2-((反)-2-((反)-6-氧-3-((Z)-2-(5-氰基苯并噻唑-2(3H)-基)乙烯基)环己-1,4-二烯基)乙烯基-3-(5-氰基)苯并噻唑盐(化合物IX)
量取30mL乙酸放入容器中，将2mmol的第一步反应得到的5-氰基-2,3-二甲基苯并噻唑季铵盐(结构见反应式III左侧)、0.67mmol的4-羟基间苯二甲醛以及2.2mmol的乙酸钠加入到容器中。在氮气保护及温度为120℃的条件下搅拌反应12小时。
将反应后的混合物过滤，并用乙腈洗涤滤饼，收集滤饼得粗产物。
将上述粗产物经过10mL乙腈反复打浆三次后得到约0.37mmol的棕色黑色固体，该棕色黑色固体粉末即为2-((反)-2-((反)-6-氧-3-((Z)-2-(5-氰基苯并噻唑-2(3H)-基)乙烯基)环己-1,4-二烯基)乙烯基-3-(5-氰基)苯并噻唑盐(化合物IX)，反应式III的产率约为52％。
对产物进行核磁共振测试，结果如下。
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).
经验证，测试结果符合结构式9的结构。
实施例7染料的特异性评价
测定染料化合物II随小牛胸腺DNA及RNA浓度增大的荧光强度变化图以及最大荧光发射峰强度与小牛胸腺DNA及RNA浓度线性关系图，来评价染料的特异性。
配置一定浓度的小牛胸腺DNA的水溶液，通过紫外吸收分光光度计测定其260nm处的吸光度值，标定其浓度为1.8mM。取100μL已标定为1.8mM的小牛胸腺DNA，加入290μL水后的稀释为0.5mM的小牛胸腺DNA水溶液。配置浓度为1mM的化合物B的DMSO(二甲基亚砜)溶液，取1.5μL，再加M-60LN溶血剂(迈瑞)至3mL，置于比色皿中，测定其荧光强度。随后，每次取0.6μL 0.5mM的小牛胸腺DNA水溶液于比色皿中，并将缓冲液搅拌均匀后，放置37℃环境中静置3min后，测定其荧光强度。最终，比色皿中小牛胸腺DNA的浓度为1μM。取每个小牛胸腺DNA浓度的最大荧光发射峰处强度，作荧光强度与小牛胸腺DNA浓度的线性关系图。RNA的浓度与荧光强度线性关系实验也按照上述步骤进行。所用仪器为紫外可见分光光度计，型号：Hp8453；荧光分光光度计，型号：FP-6500。
图20显示了化合物II随DNA浓度增加，荧光光谱的变化情况。图21显示了化合物II随RNA浓度增加，荧光光谱的变化情况。图22为荧光强度与小牛胸腺DNA和RNA浓度的线性关系图。
从图中可以看出，化合物II与DNA有浓度依赖关系，而与RNA没有浓度依赖关系，说明化合物II可以与DNA特异结合。
实施例8激光显微镜下观察化合物II对HepG2活细胞的染色
加配有化合物II、浓度为1mM的PBS缓冲液10μL于培养好HepG2细胞的六孔板中，在37℃、5％CO2的细胞培养箱中孵育30min。然后，PBS震荡清洗3次，再加入细胞培养基，共聚焦激光扫描显微镜观察细胞形态。所用仪器型号：FV1000IX81，Japan。
图23显示了观察结果。中间图为化合物II对HepG2活细胞染色的白场显微照片，左图是化合物II对HepG2活细胞染色的荧光显微照片，右图为明场图与荧光图像的叠加。如图可观察到化合物II对HepG2细胞核清晰染色，说明染料的通透性好，染核酸能力强。
实施例9激光显微镜下观察化合物III对HepG2活细胞的染色
加配有化合物III、浓度为1mM的PBS缓冲液10μL于培养好HepG2细胞的六孔板中，在37℃、5％CO2的细胞培养箱中孵育30min。然后，PBS震荡清洗3次，再加入细胞培养基，共聚焦激光扫描显微镜观察细胞形态。所用仪器型号：FV1000IX81，Japan。
图24显示了观察结果。中间图为化合物III对HepG2活细胞染色的白场显微照片，左图是化合物III对HepG2活细胞染色的荧光显微照片，右图为明场图与荧光图像的叠加。如图可观察到化合物III对HepG2细胞核清晰染色，说明染料的通透性好，染核酸能力强。
实施例10染料稳定性评价
染料用于商品化细胞染料剂，需具有一定的高温稳定性，现有染料由于高温稳定性较差，在实际商业化应用中受到一定的限制。为了改善该情况，本发明从分子结构角度设计，开发多种新型染料，以期提高染料稳定性。
为了评价高温稳定性，将50mg/L的不同染料/乙二醇溶液置于50℃恒温箱中，于不同时间取0.5ml测试其染料浓度，根据不同时间点染料浓度绘制降解曲线。测试的染料为：文献(Nucleic Acids Research,2015,Vol.43,No.18 8651–8663)报道的染料QCy-DT、染料5(化合物V)、染料6(化合物VI)、染料9(化合物IX)。所用仪器为UV- VIS紫外可见分光光度计，型号：TCC-240A,SHIMADZU,Japan。
图25为降解曲线，四条曲线从上至下依次对应于染料5、染料6、染料9、染料QCy-DT。从图中可以看出，染料QCy-DT降解的程度最严重，而染料5、染料6、染料9的降解情况较轻微，说明在化合物R3处引入吸电子基团能一定程度提高化合物的稳定性，有利于化合物血细胞染色等商业化应用。
实施例11染料细胞穿透力评价
染料用于商品化细胞染料剂，需具有较好的细胞穿透力，现有染料由于分子结构logP值(化合物在正辛醇和水中的分配系数比值的对数值)较小，对活细胞的穿透能力较弱，在实际商业化应用中受到一定的限制。为了改善该状况，本发明从分子结构角度设计，引入多种化学基团，在提高染料稳定性的同时，提升染料分子对细胞的穿透力。
为了验证细胞穿透能力，将不同染料化合物分别配制为浓度1mM的PBS缓冲液10μL加至培养好的HepG2细胞的12孔板中，在37℃、5％CO2的细胞培养箱中孵育。然后，在不同时间点，用PBS震荡清洗3次，再加入细胞培养基，采用多功能酶标仪(Thermo,USA)测定各孔道荧光光度。
图26显示了测试结果，四条曲线从上至下依次对应于染料6、染料9、染料5、染料QCy-DT。如图所示，染料5、6、9对细胞的穿透力要优于染料QCy-DT。
实施例12本发明的花菁染料在采用流式细胞术识别待测血液样本的微生物中的用途
首先按照如下配方配制染料试剂A1和溶血剂B1。

其中，第一染料包括具有上述结构式1的化合物并且用于对微生物进行染色，第二染料包括具有如下化学式的化合物并且用于对细胞进行染色：
然后，将20微升的染料试剂A1、1毫升的溶血剂B1与20微升的经抗凝处理的含有克雷伯氏假单孢菌的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、侧向散射光强度SSC、第一荧光强度FL1和第二荧光强度FL2；根据前向散射光光强信息和第一荧光强度生成如图27A所示的第一散点图，并且根据侧向散射光光强信息和第二荧光强度生成如图27B所示的第二散点图；基于图27A所示的第一散点图能够识别出在待测血液样本中存在微生物，并且基于图27B所示的第二散点图能够对待测血液样本中的白细胞进行四分类，得到如表1所示的白细胞分类结果。
在现有的血液分析仪(迈瑞，型号为BC-6800)上采用DIFF通道(使用迈瑞BC-6800的DIFF配套试剂)对同一支待测血液样本进行测试，得到如表1所示的白细胞分类结果。
表1白细胞分类结果
从表1可以看出，按照本发明获得的白细胞分类结果与按照现有的BC-6800获得的白细胞分类结果基本一致。由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的微生物检测和白细胞检测。
实施例13 本发明的花菁染料在采用流式细胞术识别待测模拟体液样本的微生物中的用途
将一定量的克雷伯氏假单孢菌加入到生理盐水中，从而得到待测的模拟体液样本；采用实施例12的试剂和方法对上述待测的模拟体液样本进行测试，以获得前向散射光强度FSC和第一荧光强度FL1；根据前向散射光光强信息和第一荧光强度生成如图28所示的第一散点图；基于图28所示的第一散点图能够识别出在模拟体液样本中存在微生物。
实施例14 本发明的花菁染料在采用流式细胞术识别待测血液样本的微生物中的用途
首先按照如下配方配制染料试剂A2和溶血剂B2。


其中，第一染料包括具有上述结构式2的化合物并且用于对微生物进行染色，第二染料包括具有如下化学式的化合物并且用于对细胞进行染色：
然后，将20微升的染料试剂A2、1毫升的溶血剂B2与20微升的经抗凝处理的含有克雷伯氏假单孢菌的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、侧向散射光强度SSC、第一荧光强度FL1和第二荧光强度FL2；根据前向散射光光强信息和第一荧光强度生成如图29A所示的第一散点图，并且根据侧向散射光光强信息和第二荧光强度生成如图29B所示的第二散点图；基于图29A所示的第一散点图能够识别出在待测血液样本中存在微生物，并且基于图29B所示的第二散点图能够对待测血液样本中的白细胞进行四分类，得到如表2所示的白细胞分类结果。
在现有的血液分析仪(迈瑞，型号为BC-6800)上采用DIFF通道(使用迈瑞BC-6800的DIFF配套试剂)对同一支待测血液样本进行测试，得到如表2所示的白细胞分类结果。
表2白细胞分类结果

从表2可以看出，按照本发明获得的白细胞分类结果与按照现有的BC-6800获得的白细胞分类结果基本一致。由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的微生物检测和白细胞检测。
实施例15本发明的花菁染料在采用流式细胞术识别待测模拟体液样本的微生物中的用途
将一定量的克雷伯氏假单孢菌加入到生理盐水中，从而得到待测的模拟体液样本；采用实施例14的试剂和方法对上述待测的模拟体液样本进行测试，以获得前向散射光强度FSC和第一荧光强度FL1；根据前向散射光光强信息和第一荧光强度生成如图30所示的第一散点图；基于图11所示的第一散点图能够识别出在模拟体液样本中存在微生物。
实施例16本发明的花菁染料在采用流式细胞术识别待测血液样本的微生物中的用途
首先按照如下配方配制染料试剂A3和溶血剂B3。

其中，第一染料包括具有上述结构式3的化合物并且用于对微生物进行染色，第二染料包括具有如下化学式的化合物并且用于对细胞进行染色：
然后，将20微升的染料试剂A3、1毫升的溶血剂B3与20微升的经抗凝处理的含有大肠杆菌的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、侧向散射光强度SSC、第一荧光强度FL1和第二荧光强度FL2；根据前向散射光光强信息和第一荧光强度生成如图31A所示的第一散点图，并且根据侧向散射光光强信息和第二荧光强度生成如图31B所示的第二散点图；基于图31A所示的第一散点图能够识别出在待测血液样本中存在微生物，并且基于图31B所示的第二散点图能够对待测血液样本中的白细胞进行四分类，得到如表3所示的白细胞分类结果。
在现有的血液分析仪(迈瑞，型号为BC-6800)上采用DIFF通道(使用迈瑞BC-6800的DIFF配套试剂)对同一支待测血液样本进行测试，得到如表3所示的白细胞分类结果。
表3白细胞分类结果
从表3可以看出，按照本发明获得的白细胞分类结果与按照现有的BC-6800获得的白细胞分类结果基本一致。由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的微生物检测和白细胞检测。
实施例17本发明的花菁染料在采用流式细胞术识别待测的模拟体液样本的微生物中的用途
将一定量的大肠杆菌加入到生理盐水中，从而得到待测的模拟体液样本；采用实施例16的试剂和方法对上述待测的模拟体液样本进行测试，以获得前向散射光强度FSC和第一荧光强度FL1；根据前向散射光光强信息和第一荧光强度生成如图32所示的第一散点图；基于图32所示的第一散点图能够识别出在模拟体液样本中存在微生物。
实施例18本发明的花菁染料在采用流式细胞术识别待测血液样本的微生物中的用途
首先按照如下配方配制染料试剂A4和溶血剂B4。


其中，第一染料包括具有上述结构式1的化合物并且用于对微生物进行染色，第二染料包括具有如下化学式的化合物并且用于对细胞进行染色：
然后，将20微升的染料试剂A4、1毫升的溶血剂B4与20微升的经抗凝处理的含有克雷伯氏假单孢菌的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、第一荧光强度FL1和第二荧光强度FL2；根据前向散射光光强信息和第一荧光强度生成如图33A所示的第一散点图，并且根据前向散射光光强信息和第二荧光强度生成如图33B所示的第二散点图；基于图33A所示的第一散点图能够识别出在待测血液样本中存在微生物，并且基于图33B所示的第二散点图能够识别待测血液样本中的白细胞、有核红细胞和嗜碱性粒细胞并对其进行计数。
由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的微生物检测和有核红细胞检测。
实施例19本发明的花菁染料在采用流式细胞术识别待测模拟体液样本的微生物中的用途
将一定量的克雷伯氏假单孢菌加入到生理盐水中，从而得到模拟的待测体液样本；采 用实施例18的试剂和方法对上述待测的模拟体液样本进行测试，以获得前向散射光强度FSC和第一荧光强度FL1；根据前向散射光光强信息和第一荧光强度生成如图34所示的第一散点图；基于图34所示的第一散点图能够识别出在模拟体液样本中存在微生物。
实施例20本发明的花菁染料在采用流式细胞术识别待测血液样本的微生物中的用途
首先按照如下配方配制染料试剂A5和溶血剂B5。

其中，第一染料包括具有上述结构式3的化合物并且用于对微生物进行染色，第二染料包括具有如下化学式的化合物并且用于对细胞进行染色：
然后，将20微升的染料试剂A5、1毫升的溶血剂B5与20微升的经抗凝处理的含有克雷伯氏假单孢菌的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、第一荧光强度FL1和第二荧光强度FL2；根 据前向散射光光强信息和第一荧光强度生成如图35A所示的第一散点图，并且根据前向散射光光强信息和第二荧光强度生成如图35B所示的第二散点图；基于图35A所示的第一散点图能够识别出在待测血液样本中存在微生物，并且基于图35B所示的第二散点图能够识别待测血液样本中的白细胞、有核红细胞和嗜碱性粒细胞并对其进行计数。
由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的微生物检测和有核红细胞检测。
实施例21本发明的花菁染料在采用流式细胞术识别待测的模拟体液样本的微生物中的用途
将一定量的克雷伯氏假单孢菌加入到生理盐水中，从而得到待测的模拟体液样本；采用实施例20的试剂和方法对上述待测的模拟体液样本进行测试，以获得前向散射光强度FSC和第一荧光强度FL1；根据前向散射光光强信息和第一荧光强度生成如图36所示的第一散点图；基于图36所示的第一散点图能够识别出在模拟体液样本中存在微生物。
实施例22本发明的花菁染料在采用流式细胞术识别待测血液样本的寄生虫中的用途
将20微升的染料试剂A4(实施例18，第一染料用于对寄生虫进行染色)、1毫升的溶血剂B4(实施例18)与20微升的经抗凝处理的感染了疟疾的患者的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、第一荧光强度FL1和第二荧光强度FL2；根据前向散射光光强信息和第一荧光强度生成如图37A所示的第一散点图，并且根据前向散射光光强信息和第二荧光强度生成如图37B所示的第二散点图；基于图37A所示的第一散点图能够识别出在待测血液样本中存在疟原虫，并且基于图37B所示的第二散点图能够识别待测血液样本中的白细胞、有核红细胞和嗜碱性粒细胞并对其进行计数。
由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的寄生虫检测和有核红细胞检测。
实施例23本发明的花菁染料在采用流式细胞术识别待测血液样本的寄生虫中的用途
首先按照如下配方配制染料试剂A6和溶血剂B6。


其中，第一染料包括具有上述结构式2的化合物并且用于对寄生虫进行染色，第二染料包括具有如下化学式的化合物并且用于对细胞进行染色：
然后，将20微升的染料试剂A6、1毫升的溶血剂B6与20微升的经抗凝处理的感染了疟疾的患者的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、第一荧光强度FL1和第二荧光强度FL2；根据前向散射光光强信息和第一荧光强度生成如图38A所示的第一散点图，并且根据前向散射光光强信息和第二荧光强度生成如图38B所示的第二散点图；基于图38A所示的第一散点图能够识别出在待测血液样本中存在疟原虫，并且基于图38B所示的第二散点图能够识别待测血液样本中的白细胞、有核红细胞和嗜碱性粒细胞并对其进行计数。
由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的寄生虫检测和有核红细胞检测。
实施例24本发明的花菁染料在采用流式细胞术识别待测血液样本的寄生虫中的用途
将20微升的染料试剂A5(实施例20，第一染料用于对寄生虫进行染色)、1毫升的溶血剂B5(实施例20)与20微升的经抗凝处理的感染了疟疾的患者的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、第一荧光强度FL1和第二荧光强度FL2；根据前向散射光光强信息和第一荧光强度生成如图39A所示的第一散点图，并且根据前向散射光光强信息和第二荧光强度生成如图39B所示的第二散点图；基于图39A所示的第一散点图能够识别出在待测血液样本中存在疟原虫，并且基于图39B所示的第二散点图能够识别待测血液样本中的白细胞、有核红细胞和嗜碱性粒细胞并对其进行计数。
由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的寄生虫检测和有核红细胞检测。
实施例25本发明的花菁染料在采用流式细胞术识别待测血液样本的寄生虫中的用途
将20微升的染料试剂A2(实施例14，第一染料用于对寄生虫进行染色)、1毫升的溶血剂B2(实施例14)与20微升的经抗凝处理的感染了疟疾的患者的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、侧向散射光强度SSC、第一荧光强度FL1和第二荧光强度FL2；根据前向散射光光强信息和第一荧光强度生成如图40A所示的第一散点图，并且根据侧向散射光光强信息和第二荧光强度生成如图40B所示的第二散点图；基于图40A所示的第一散点图能够识别出在待测血液样本中存在疟原虫，并且基于图40B所示的第二散点图能够对待测血液样本中的白细胞进行四分类，得到如表4所示的白细胞分类结果。
在现有的血液分析仪(迈瑞，型号为BC-6800)上采用DIFF通道(使用迈瑞BC-6800的DIFF配套试剂)对同一支待测血液样本进行测试，得到如表4所示的白细胞分类结果。
表4白细胞分类结果
从表4可以看出，按照本发明获得的白细胞分类结果与按照现有的BC-6800获得的白细胞分类结果基本一致。由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的寄生虫检测和白细胞检测。
实施例26本发明的花菁染料在采用流式细胞术识别待测血液样本的寄生虫中的用途
首先按照如下配方配制染料试剂A7和溶血剂B7。


其中，第一染料包括具有上述表1中的结构式3的化合物并且用于对寄生虫进行染色，第二染料包括具有如下化学式的化合物并且用于对细胞进行染色：
然后，将20微升的染料试剂A7、1毫升的溶血剂B7与20微升的经抗凝处理的感染了疟疾的患者的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、侧向散射光强度SSC、第一荧光强度FL1和第二荧光强度FL2；根据前向散射光光强信息和第一荧光强度生成如图41A所示的第一散点图，并且根据侧向散射光光强信息和第二荧光强度生成如图41B所示的第二散点图；基于图41A所示的第一散点图能够识别出在待测血液样本中存在疟原虫，并且基于图41B所示的第二散点图能够对待测血液样本中的白细胞进行四分类，得到如表5所示的白细胞分类结果。
在现有的血液分析仪(迈瑞，型号为BC-6800)上采用DIFF通道(使用迈瑞BC-6800的DIFF配套试剂)对同一支待测血液样本进行测试，得到如表5所示的白细胞分类结果。
表5白细胞分类结果
从表5可以看出，按照本发明获得的白细胞分类结果与按照现有的BC-6800获得的白细胞分类结果基本一致。由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的寄生虫检测和白细胞检测。
实施例27本发明的花菁染料在采用流式细胞术识别待测血液样本的寄生虫中的用途
首先按照如下配方配制染料试剂A8和溶血剂B8。


其中，第一染料包括具有上述结构式4的化合物并且用于对寄生虫进行染色，第二染料包括具有如下化学式的化合物并且用于对细胞进行染色：
然后，将20微升的染料试剂A8、1毫升的溶血剂B8与20微升的经抗凝处理的感染了疟疾的患者的待测血液样本混合，使混合物在42℃条件下孵育30秒，形成测定用的待测样本液；接着，使用具有激发波长约为450nm的蓝色激光器的流式分析仪对待测样本液进行测试，以获得前向散射光强度FSC、侧向散射光强度SSC、第一荧光强度FL1和第二荧光强度FL2；根据前向散射光光强信息和第一荧光强度生成如图42A所示的第一散点图，并且根据侧向散射光光强信息和第二荧光强度生成如图42B所示的第二散点图；基于图42A所示的第一散点图能够识别出在待测血液样本中存在疟原虫，并且基于图42B所示的第二散点图能够对待测血液样本中的白细胞进行四分类，得到如表6所示的白细胞分类结果。
在现有的血液分析仪(迈瑞，型号为BC-6800)上采用DIFF通道(使用迈瑞BC-6800的DIFF配套试剂)对同一支待测血液样本进行测试，得到如表6所示的白细胞分类结果。
表6白细胞分类结果

从表6可以看出，按照本发明获得的白细胞分类结果与按照现有的BC-6800获得的白细胞分类结果基本一致。由此可见，本发明实施例能够通过对同一份待测血液样本的一次检测同时实现血液中的寄生虫检测和白细胞检测。
以上在说明书、附图以及权利要求书中提及的特征或者特征组合，只要在本发明的范围内是有意义的并且不会相互矛盾，均可以任意相互组合使用或者单独使用。针对本发明提供的样本分析方法所说明的优点和特征以相应的方式适用于本发明提供的样本分析仪和DNA特异性染料的应用，反之亦然。
以上所述仅为本发明的优选实施例，并非因此限制本发明的专利范围，凡是在本发明的发明构思下，利用本发明说明书及附图内容所作的等效变换，或直接/间接运用在其他相关的技术领域均包括在本发明的专利保护范围内。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (27)

  1. 具有如通式I所示结构的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，
    其中，
    R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基；
    R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基；
    Y不存在或为抗衡阴离子；
    并且限定：
    当R3为氢时，R1和R2不同时为甲基且R1和R2不同时为苄基。
  2. 如权利要求1所述的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，其中，R1和R2相同或不同，并且独立地选自C1-6直链烷基、C1-6直链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基。
  3. 如权利要求1或2所述的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，其中，R1和R2中的至少一个为C1-18直链或支链亚烷基-磺酸基。
  4. 如权利要求1-3任一项所述的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，其中，R1和R2不同且独立地选自C1-6直链烷基、苄基、C1-6直链亚烷基-羧基、C1-6直链亚烷基-磺酸基、C1-6直链亚烷基-巯基、C1-6直链亚烷基-氨基。
  5. 如权利要求1-3任一项所述的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，其中，R1和R2相同且选自C1-6直链烷基、C1-6直链亚烷基-磺酸基、C1-6直链亚烷基-羧基。
  6. 如权利要求1-5任一项所述的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，其中，R3选自氢、磺酸基、卤素、氰基、C1-6烷基。
  7. 如权利要求1-6任一项所述的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，其中，当Y为抗衡阴离子时，Y选自卤素离子(例如F-、Cl-、Br-、I-)、ClO4-、PF6-、CF3SO3-、BF4-、乙酸根、甲磺酸根或对甲苯磺酸根。
  8. 如权利要求1-7任一项所述的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，其中，当Y不存在时，所述化合物为内盐。
  9. 如权利要求8所述的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，其中，R1和R2中的至少一个选自C1-18直链或支链亚烷基-磺酸基、C1-18直链或支链亚烷基-羧基。
  10. 如权利要求1-9任一项所述的化合物、其水合物、溶剂化物、立体异构体、互变异构体或晶型，所述化合物具有以下任一结构：

  11. 如权利要求1-9任一项所述的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型，所述化合物具有以下任一结构：
  12. 一种缀合物，所述缀合物包含权利要求1-11中任一项化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型。
  13. 一种用于生物样品染色的组合物，其中所述组合物包含权利要求1-11中任一项的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型或权利要求12的缀合物。
  14. 权利要求13的组合物，其中所述生物样品为核酸，优选为脱氧核糖核酸。
  15. 权利要求1-11中任一项的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型、权利要求12的缀合物或权利要求13的组合物在对生物样品进行染色中的用途。
  16. 权利要求15的用途，其中所述生物样品为核酸，优选为脱氧核糖核酸。
  17. 权利要求1-11中任一项的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型、权利要求12的缀合物或权利要求13的组合物在采用流式细胞术识别待测血液样本中的寄生虫中的用途。
  18. 权利要求1-11中任一项的化合物或其水合物、溶剂化物、立体异构体、互变异构体或晶型、权利要求12的缀合物或权利要求13的组合物在采用流式细胞术识别待测血液样本或待测体液样本中的微生物中的用途。
  19. 一种样本分析方法，其特征在于，所述样本分析方法包括下列步骤：
    采用包含第一染料的染料试剂和用于溶解红细胞的溶血剂处理同一份待测生物样本，以获得待测样本液，其中，所述待测生物样本为待测血液样本或待测体液样本，所述第一染料能对微生物进行染色；
    使所述待测样本液中的粒子逐个通过光学检测区并用光照射流过所述光学检测区的粒子，以获得所述待测样本液中的粒子在被光照射之后所产生的散射光信息和荧光信息，所述荧光信息包括来自所述第一染料的第一荧光信息；以及
    基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物。
  20. 一种样本分析仪，其特征在于，所述样本分析仪包括：
    采样装置，用于定量吸取待测生物样本，其中，所述待测生物样本为待测血液样本或待测体液样本；
    样本制备装置，具有反应池和试剂供应部，其中，所述反应池用于接收所述采样装置所吸取的所述待测生物样本，以及接收所述试剂供应部提供的包含第一染料的染料试剂和用于溶解红细胞的溶血剂，所述采样装置所吸取的所述待测生物样本与所述试剂供应部提供的染料试剂和溶血剂在所述反应池中混合，以制备成待测样本液，其中，所述第一染料能对微生物进行染色；
    光学检测装置，包括光源、流动室、散射光检测器和荧光检测器，所述光源用于发射光束以照射所述流动室，所述流动室与所述反应池连通并且所述待测样本液中的粒子可逐个通过所述流动室，所述散射光检测器用于检测通过所述流动室的粒子在被光照射后产生的散射光信息，所述荧光检测器用于检测通过所述流动室的粒子在被光照射后产生的荧光信息，所述荧光信息包括来自所述第一染料的第一荧光信息；以及
    处理器，被配置为从所述光学检测装置获取所述散射光信息和所述荧光信息，并且基于所述散射光信息和所述第一荧光信息识别待测样本液中的微生物。
  21. 如权利要求20所述的样本分析仪，其特征在于，所述处理器还被配置为在基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的微生物时执行下列步骤：
    基于所述散射光信息、优选前向散射光信息和所述第一荧光信息生成第一散点图；并且
    基于所述第一散点图识别所述待测样本液中的微生物。
  22. 如权利要求21所述的样本分析仪，其特征在于，所述处理器还被配置为：
    从所述第一散点图获取微生物特征区域和白细胞区域，所述微生物特征区域的第一荧光信息的强度大于所述白细胞区域的第一荧光信息的强度；并且
    基于所述微生物特征区域识别所述待测样本液中的微生物。
  23. 如权利要求20-22中任一项所述的样本分析仪，其特征在于，所述第一染料为能与脱氧核糖核酸特异性结合的染料，尤其是所述第一染料包括如权利要求1-11中任一项所述的化合物。
  24. 一种血液分析方法，其特征在于，所述血液分析方法包括下列步骤：
    采用包含第一染料的染料试剂和用于溶解红细胞的溶血剂处理同一份待测血液样本，以获得待测样本液，其中，所述第一染料能对血液中的寄生虫进行染色，并且所述第一染料并且所述第一染料包括具有通式Ⅰ的结构的化合物，尤其是包括如权利要求1-11中任一项所述的化合物；
    其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基，R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基，Y不存在或为抗衡阴离子；
    使所述待测样本液中的粒子逐个通过光学检测区并用光照射流过所述光学检测区的粒子，以获得所述待测样本液中的粒子在被光照射之后所产生的散射光信息和荧光信息，所述荧光信息包括来自所述第一染料的第一荧光信息；以及
    基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫。
  25. 一种血液分析仪，其特征在于，所述血液分析仪包括：
    采样装置，用于定量吸取待测血液样本；
    样本制备装置，具有反应池和试剂供应部，其中，所述反应池用于接收所述采样装置所吸取的所述待测血液样本，以及接收所述试剂供应部提供的包含第一染料的染料试剂和用于溶解红细胞的溶血剂，所述采样装置所吸取的所述待测血液样本与所述试剂供应部提供的染料试剂和溶血剂在所述反应池中混合，以制备成待测样本液，其中，所述第一染料包括具有通式Ⅰ的结构的化合物，尤其是包括如权利要求1-11中任一项所述的化合物；
    其中，R1和R2相同或不同，并且独立地选自C1-18直链或支链烷基、C1-18直链或支链亚烷基-M，M选自磺酸基、苯基、羧基、巯基、氨基，R3选自氢、磺酸基、卤素、氰基、C1-6烷基、羟基、C1-6烷氧基、卤代C1-6烷基，Y不存在或为抗衡阴离子；
    光学检测装置，包括光源、流动室、散射光检测器和荧光检测器，所述光源用于发射光束以照射所述流动室，所述流动室与所述反应池连通并且所述待测样本液中的粒子可逐个通过所述流动室，所述散射光检测器用于检测通过所述流动室的粒子在被光照射后产生的散射光信息，所述荧光检测器用于检测通过所述流动室的粒子在被光照射后产生的荧光信息，所述荧光信息包括来自所述第一染料的第一荧光信息；以及
    处理器，被配置为从所述光学检测装置获取所述散射光信息和所述荧光信息，并且基于所述散射光信息和所述第一荧光信息识别待测样本液中的寄生虫。
  26. 如权利要求25所述的血液分析仪，其特征在于，所述处理器还被配置为在基于所述散射光信息和所述第一荧光信息识别所述待测样本液中的寄生虫时执行下列步骤：
    基于所述散射光信息、优选前向散射光信息和所述第一荧光信息生成第一散点图；并且
    基于所述第一散点图识别所述待测样本液中的寄生虫。
  27. 如权利要求26所述的血液分析仪，其特征在于，所述处理器还被配置为：
    从所述第一散点图获取寄生虫特征区域和白细胞区域，所述寄生虫特征区域的第一荧光信息的强度大于所述白细胞区域的第一荧光信息的强度；并且
    基于所述寄生虫特征区域识别所述待测样本液中的寄生虫。
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